Metabolomic profiling reveals effects of marein on energy metabolism in HepG2 cells

Background

Type 2 Diabetes Mellitus (T2DM) is associated with a sedentary lifestyle and obesity. It is a consequence of insulin resistance (IR) unlike type 1 which is an autoimmune disease, but both types lead to hyperglycemia [1]. A variety of pathological conditions are directly associated with T2DM including renal, cardiovascular and hepatic diseases [2]. The liver, as a central metabolic organ, plays a key role in regulating glucose homeostasis and lipid metabolism, responding to changes in substrate and hormone levels such as insulin and glucagon. As such, functional hepatic overload is common in the development of metabolic diseases such as non-alcoholic fatty liver disease (NAFLD) and T2DM [3]. Thus, tracing effective treatments targeting the causes and consequences of T2DM is extremely important. 

Despite the fact that many drugs have been approved by international agencies to treat the consequences of T2DM and IR, harmful side-effects and elevated costs could impair therapeutic adherence. Therefore, a therapeutic scheme involving lifestyle modifications, pharmacological treatment and phytochemicals may be more effective in the treatment of metabolic diseases [4]. In this sense, metabolomics and other omic technologies have great potential to drive T2DM research as well as potential therapeutic interventions due to their role in identifying and quantifying biomolecules or cellular processes involved in pathophysiological conditions through illustration of genetic status [5]. 

This preprint investigates the effects of the Coreopsis tinctoria phytoproducts on liver energy metabolism as a potential therapeutic intervention for T2DM.

 

Key findings

Beneficial effects of the ethyl acetate extract of Coreopsis tinctoria (AC) on serum and body parameters and liver structure in an animal model

The results show that the high-fat diet (HFD) was effective in promoting obesity and metabolic dysregulation in rats as demonstrated by elevated cholesterol, triglycerides and lipoproteins. HFD also impaired glucose homeostasis, increasing blood glucose and insulinemia which, as expected, decreases the insulin sensitivity as measured by the ITT assay. Interestingly, the well known anti-diabetic and anti-obesity effects of metformin were confirmed in the HFD model presented here. Strikingly, eight weeks of treatment with the ethyl acetate extract (AC) of Coreopsis tinctoria reduced all parameters altered by HFD similarly to metformin. The observed effects of AC were associated with an overall improvement of dyslipidemia and IR. Histological analysis showed a steatotic pattern in the HFD group that was recovered more efficiently by metformin treatment than by AC treatment. 

Marein improves glucose metabolism in HepG2 cells

The results presented in this preprint show that exposure to high glucose concentrations (HG) reduced glucose uptake in a HepG2 cell line. This effect was dependent on time and glucose concentration. Marein improved glucose uptake at a concentration of 5 µM in HepG2 cells, and restored glycogen levels and hexokinase (HK) activity, which are important parameters of normal glucose metabolism. Cell survival after exposure to marein was not affected even at the high concentration of 40 μM, although the methodological assay of cell survival was not described by the authors. Protein and mRNA expression of the key enzymes of the gluconeogenesis pathway (PEPCK and G6Pase) were evaluated and although representative blots were overexposed, HG treatment increased the expression of both enzymes and marein was effective in restoring these expressions.

Metabolic changes of HepG2 cells by high glucose are reverted by Marein

The metabolic profile of HepG2 cells exposed to HG medium was quite different from control cells, suggesting that excessive glucose promotes metabolic disturbances. Interestingly, marein treatment of HG cells shifts  the metabolic profile towards the control suggesting a reversal of the hyperglycemic damage associated with HG. 

HG impairs glucose metabolism shifting towards glycolysis and marein reverses this by restoring the levels of TCA enzymes

Evaluation of the tricarboxylic acid cycle (TCA) cycle and glycolysis metabolites by the authors demonstrated a reduction in DHAP and 3-phosphoglycerate levels, as well as an increase in cis-aconitate, succinate and malate levels, which was modulated and prevented by marein. Interestingly, the expression of TCA cycle enzymes including succinate dehydrogenase, aconitase and citrate synthase was increased by HG, an effect that was reversed by marein. Finally, the authors observed higher levels of ATP, pyruvate and lactate which, together with reduced glucose uptake, suggests increased gluconeogenesis, a metabolic hallmark of insulin resistance. The preventive effect of marein in the context of IR illustrates its potential therapeutic effect against T2DM. 

Figure 1: Illustration of the TCA. This preprint quantified the metabolites analyzed in the different experimental groups. The results showed that cells exposed to the high glucose medium had an almost opposite metabolomic characteristic compared to control cells. Cells exposed marein (5 and 10 μM) showed an intermediate pattern in a situation of high glucose. 

 

Why I think this preprint is important

This preprint investigates the effect of phytochemicals derived from Coreopsis tinctoria on energy metabolism in the context of high glucose exposure. The observed effects suggest that phytochemicals derived from C. tinctoria exhibit preventive effects against the metabolic dysregulation caused by high glucose exposure – it therefore proposes that marein could be used as a potential therapeutic intervention for T2DM and other metabolic diseases.

The fact that the preprint has not been published and has been on BioRxiv since 2017 could perhaps be due to some methodological gaps, analyzed in the “Questions and Suggestions” section below. Furthermore, the preprint authors did publish 2 articles linking the beneficial effects of marein exposure to insulin resistance. The first, published in 2016, investigated the effects of marein on the PI3K/AKT and AMPK pathways in HepG2 cells, and showed that marein contributed to alleviating insulin resistance induced by high glucose levels. The second, published in 2018, investigated the effects of C. tinctoria tea on obesity induced by a high-fat diet in rats, and the results demonstrated that the tea promoted changes in insulin-related genes at the level of the transcriptome.

 

Questions and suggestions for the authors

1. The authors could describe in more detail the route of administration of AC, as well as provide more details regarding the animal sex, age, and housekeeping conditions (temperature, light exposure, humidity). Given that the authors carried out a whole-body metabolic assessment, the food intake should be measured for each animal group. As a suggestion, the authors could consider performing an oral glucose tolerance test (OGTT) to strengthen the results on ITT. 
2. The animal groups were described as Control Diet (CD), High-fat Diet (HFD), Metformin and ethyl acetate extract of Coreopsis tinctoria (AC) in three doses. However, the authors did not consider the possibility that the vehicles of metformin and AC could play a role in the phenotypes observed. This is critical considering that metformin and AC vehicles are distinct and could modulate the parameters assessed. As a suggestion, the authors could consider the inclusion of groups of CD or HFD animals that received only the drug dilution vehicle to clarify that the observed effects were not due to drug vehicles. 
3. The authors should consider describing the methodology for measuring HK activity and glycogen, as well as adding a description of other experiments. This includes providing information on the kits used for the serum quantifications, references to the ITT and histopathological examination. Also, the authors’ assessment of glucose uptake by the 2-NBDG analogue could help to describe glucose metabolism. In addition, the authors should revise the references in the methods section, as they do not match those in the reference list. 
4. The three-dimensional PCA score plot with clustering of each group (control, HG, metformin and marein) was used in the statistical analyses to compare metabolic patterns between the groups. However, the exact meaning of the principal components 1, 2 and 3 in figures 4F and supplemental figures were not described or present. A slight, but important, detail is that authors should consider the standardization of color patterns of experimental groups to improve data presentation. A minor issue is that figure 6 methods were not described and its formatting should be improved. Also, the authors could consider providing the full western blotting membranes as supplementary figures.
5. How do the authors determine the lipid accumulation using hematoxylin& eosin staining? The authors could consider using specific staining methods to directly assess lipid accumulation, such as the Oil Red O [6]. In addition, the authors could consider changing the size representation presented on figure 2 to 50 μm rather than 50 μM.
6. Even without considering by which route the extract of C. tinctoria was introduced to the animals, the authors should consider the effects of ethyl acetate (EtAc) and its metabolites on metabolism, such as conversion to ethanol (EtOH) in vivo [7, 8]. Furthermore, evaluating the effects of C. tinctoria in a pharmaceutical form closer to the usual one and using inert solvents may make the results more applicable. This strengthens the need of control groups exposed only to the extract vehicles.
7. The authors could more clearly describe the methods used to obtain both the AC and marein from the studied plants. Details might include the description of plants’ culture conditions (soil, temperature, light and water regimen, etc) as well as how the AC and marein were prepared. [9] so that the methods are traceable and reproducible.
8. The legend of figure 1 describes that the insulin sensitivity index was measured as [ISI =1/(fasting insulin x fasting blood glucose)], which is somewhat unusual. The authors could consider the use of alternative gold-standard methods to determine insulin resistance such as HOMA-IR or QUICKI [10,11].
9. The pyruvate tolerance test (PTT) estimates hepatic gluconeogenesis by analyzing glucose release following bolus administration of pyruvate [12]. Given that the authors’ conclusion is that HG-exposed cells exhibit the most active gluconeogenesis pathway and marein prevents this effect, the authors could consider performing a pyruvate tolerance test to support this hypothesis.

 

References

[1] Olivares-Reyes, J.A.; Arellano-Plancarte, A.; Castillo-Hernandez, J.R. Angiotensin II and the development of insulin resistance: implications for diabetes. Mol Cell Endocrinol. 2009 Apr; 302(2):128-39;

[2] <https://www.who.int/news-room/fact-sheets/detail/diabetes> Accessed in: 06/13/2023;

[3] Michael, M.D. et al. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol Cell. 2000 Jul; 6(1):87-97;

[4] Osadebe, P.O.; Odoh, E.U.; Uzor, P.F. Natural Products as Potential Sources of Antidiabetic Drugs. British Journal of Pharmaceutical Research. 2014 Sep; 4:2075-2095;

[5] San-Martin, B.S. et al. Metabolomics as a potential tool for the diagnosis of growth hormone deficiency (GHD): a review. Arch Endocrinol Metab. 2021 Nov; 64(6):654-663;

[6] Rector, R.S. et al. Daily exercise increases hepatic fatty acid oxidation and prevents steatosis in Otsuka Long-Evans Tokushima Fatty rats. Am J PhysiolGastrointest Liver Physiol. 2008 Mar; 294(3):G619-26;

[7] Gallaher, E. J., & Loomis, T. A. Metabolism of Ethyl Acetate in the Rat: Hydrolysis to Ethyl Alcohol in Vitro and in Vivo. Toxicology and Applied Pharmacology. 1975 Jun; 34:309-313;

[8] Crowell, S. R. et al. Physiologically based pharmacokinetic modeling of ethyl acetate and ethanol in rodents and humans. RegulToxicolPharmacol. 2015 Oct; 73(1):452-62;

[9] Stepanova, A.N. Plant Biology Research: What Is Next? Front Plant Sci. 2021 Sep;12:749104;

[10] Bowe, J.E. et al. Metabolic phenotyping guidelines: assessing glucose homeostasis in rodent models. J Endocrinol. 2014 Sep; 222(3):G13-25;

[11] Muniyappa, R. et al. Assessing Insulin Sensitivity and Resistance in Humans. [Updated 2021 Aug 9]. In: Feingold KR, Anawalt B, Blackman MR, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK278954/;

[12] Hughey, C.C. et al. Approach to assessing determinants of glucose homeostasis in the conscious mouse. Mamm Genome. 2014 Oct; 25(9-10):522-38.

Tags: hepg2 cells, insulin-resistance, marein, metabolomics, type 2 diabetes mellitus

doi: https://doi.org/10.1242/prelights.35826

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