3.1. AAE Inhibits Cholesterogenesis in HuH7 Cells
Our previously published studies have already shown that AAE reduces total cholesterol levels both in vitro, in hepatic cultured cells, as well as in vivo, in humans affected by mild hypercholesterolemia [29
]. Our results confirm those published by other groups and presenting similar effects exerted by other polyphenolic extracts [16
]. However, whether the cholesterol lowering effect of AAE is due to i) reduced cholesterogenesis, ii) increased cholesterol excretion or iii) increased conversion of cholesterol into bile acids remains elusive. The mechanism of action hypothesized for other polyphenolic extracts cannot be simply extended to AAE, since the mechanism of action of each extract seems to strongly depend on the nature and the amount of polyphenols contained in it.
In order to monitor cholesterogenesis in AAE treated cells we performed D2
O labeling of in vitro cultured hepatoma cells [36
]. Briefly, HuH7 cells were grown in a medium supplemented with D2
O for 72 h, a time sufficient to allow incorporation of Deuterium atoms into de-novo synthesized sterols and fatty acids (FAs). After labeling, lipids were extracted and derivitized with TMS (see methods section for details) to be easily visualized and quantitated by GC-MS. Chemical species endowed with molecular masses heavier (mainly Δm/z of 2–4 Da) than those naturally occurring were found co-eluting with cholesterol and cholesterol fragments (Figure 1
a). The presence of these heavier species proves the incorporation of Deuterium in newly synthesized cholesterol and thus that cholesterogenesis was occurring in HuH7 cells.
When HuH7 cell were cultured in the presence 10 μM Simvastatin or 10 μM Atorvastatin, total intracellular cholesterol levels were reduced (Atorvatatin 0.55 ± 0.09 fold induction compared to vehicle mean ± SEM (standard error of mean), p
< 0.001; Simvastatin 0.52 ± 0.06, p
< 0.001) (Figure 1
c). Moreover, the intensity of peaks corresponding to deuterated cholesterol molecules were all decreased, confirming, as expected, their molecular mechanism of action resulting in inhibition of de-novo cholesterol synthesis (Figure 1
a,b). AAE treatment (400 mg/L) resulted in a reduction of total cholesterol levels (0.48 ± 0.05 fold, p
< 0.001) with a potency similar to that exerted by the two statins (Figure 1
c). AAE reduced as well cholesterol deuterated peaks, proving that its cholesterol lowering activity involves inhibition of de novo cholesterogenesis (Figure 1
3.3. AAE Reprograms FA Metabolism and Diverts Acetyl-CoA to Krebs Cycle
The effect exerted by AAE on intracellular cholesterol and FA levels (summarized in Figure 2
a), prompted us to further extend the metabolic profiling of AAE treated HuH7 cells looking for other metabolites altered by the treatment with the polyphenolic extract (Table 1
and Table S1
). Metabolic profiling was performed by Direct Infusion FT-ICR mass spectrometry (DI-FT-ICR-MS), which is characterized by unmatched ultra-high mass accuracy and resolution, that make it highly suitable in metabolite profiling [41
]. Metabolomic approaches are extremely useful tools for probing any change in metabolism accompanying drug treatments and provide invaluable insights in the mechanism of action of complex mixtures and phytocomplexes [48
We started our metabolic profiling looking at metabolites that represent energy source for the cell (Figure 2
b and Table 1
). In virtue of the high rate of their anabolic processes (lipogenesis, cholesterogenesis and protein synthesis) hepatocytes are highly demanding in terms of energy, and use of glucose, amino acids and FAs as energy sources. Intracellular levels of glucose were not altered by AAE (1.02 ± 0.08 fold, p
> 0.05) while glucose-6-P/fructose 6-P levels (isobaric compounds, 1.81 ± 0.20 fold, p
< 0.001) were increased by the treatment. Maltose, a by-product of glycogenolysis, was increased by AAE (1.54 ± 0.13 fold, p
< 0.001) (Figure 2
a) pointing toward AAE stimulating conversion of glycogen into glycolysis intermediates. Intracellular levels of lactic acid, the product of pyruvate reduction by Lactate Dehydrogenase, were diminished by AAE (0.22 ± 0.10 fold, p
< 0.001) (Figure 2
c), suggesting that pyruvate in AAE treated cells is mostly transported into mitochondria.
Pentose phosphate pathway (PPP) is an alternative route toward the glycolysis intermediate glyceraldehyde-3-P and its activity is necessary for reduced glutathione (GSH) and nucleotide production. The intracellular levels of PPP intermediates ribose 5-P (2.04 ± 0.26 fold, p
< 0.001), sedoheptulose (1.24 ± 0.12 fold, p
< 0.05), and sedoheptulose 7-P ( 1.93 ± 0.20 fold, p
< 0.001) were all augmented upon treatment with AAE (Figure 2
b). Their accumulation is compatible with an increased rate of PPP activity. Intracellular levels of the nucleotide cytidine (1.33 ± 0.07 fold, p
< 0.01), guanosine (1.21 ± 0.09 fold, p
< 0.05), and of inosine (1.34 ± 0.07 fold, p
< 0.01) were increased by AAE, while adenosine intracellular levels resulted to be not statistically altered by AAE (0.96 ± 0.07 fold, p
> 0.05) (Figure 2
b). Interestingly, GSH levels were reduced upon treatment with AAE (0.57 ± 0.06 fold, p
The first set of results so far described suggests that AAE stimulates glycolysis, PPP (both oxidative and non-oxidative branches of PPP) but not GSH and lactate production. In the absence of lactic fermentation, pyruvate is usually transported into mitochondria and converted in acetyl-CoA and citrate to be used into the Krebs cycle. In our metabolic profiling, citrate levels resulted to be increased in cells treated with AAE (1.86 ± 0.06 fold, p
< 0.001) (Figure 2
c), supporting the hypothesis of an increased transport of pyruvate into mitochondria. In cells like hepatocytes, where lipogenesis and cholesterogenesis occur, citrate is rapidly exported out of the mitochondria and used as substrate to produce malonyl-CoA, necessary for biosynthesis of palmitate and other FAs. Malonyl-CoA is also the precursor of HMG-CoA. Despite the increase in citrate, both FT-ICR and GC-MS profiling (Figure 1
and Figure 2
a, Table 1
) clearly indicate that cholesterogenesis and lipogenesis are decreased upon treatments with AAE. Moreover the intracellular levels of the bile acid chenodesoxycholic acid (CDCA), that for its synthesis requires cholesterol, is decreased upon treatment with AAE (0.50 ± 0.05 fold, p
< 0.001) (Figure 2
c). This further confirms: i) AAE ability to halt anabolic reactions involved in cholesterogenesis and ii) excludes stimulation of cholesterol conversion into bile acids as a likely mechanism underpinning AAE cholesterol-lowering activity.
Increased levels of citrate can also lead to an increased mitochondrial respiration. In line with this hypothesis, the Krebs cycle intermediate fumarate is also increased by AAE (1.32 ± 0.12 fold, p < 0.05).
To prove that the treatment with Annurca polyphenols was indeed increasing mitochondrial activity, we used the mitochondrial probe Mito Tracker CMX-ROS. The fluorescence emitted by this dye correlates with the membrane potential of the mitochondrial inter-membrane space. The latter, depending on the amount of protons transported by the electron transport chain, is a direct measurement of mitochondrial activity. Analysed by fluorimetry, HuH7 cells treated with AAE showed an increased mitochondrial activity compared to cells treated with vehicle (1.22 ± 0.02 fold, p
< 0.01, Figure S4
) confirming that AAE ignites mitochondrial respiration. Differently, AAE did not induce autophagy in HuH7 cells, as shown by the intracellular staining of the autophagy marker LC3B. The number and the size of LC3B positive punctated structures appeared, actually, decreased in AAE treated cells compared to vehicle treated cells (Figure S5
Pyruvate produced by glycolysis is not the only fuel for mitochondrial activity. Several intracellular metabolites can be involved in metabolic pathway igniting mitochondrial respiratory activity. Glutamine can be converted into glutamate and enter the Krebs cycle as alpha-ketoglutarate. Glutamine levels were reduced by AAE suggesting that glutamine can be indeed one of the sources of increased mitochondrial activity (0.62 ± 0.04 fold, p < 0.01). The use of glutamine for catabolic reactions rather than for anabolism would be also indirectly confirmed by its anabolic products GSH, as already shown, reduced by AAE.
Differently, the intracellular levels of other amino acids (both ketogenic and glucogenic ones) are either unaltered or increased by AAE excluding them as possible energy source for mitochondrial activity. Lysine (0.97 ± 0.12 fold, p > 0.05), histidine (1.08 ± 0.11 fold, p value > 0.05), or aspartate (1.08 ± 0.06 fold, p > 0.05) are unaltered by AAE while, on the contrary leucine (1.54 ± 0.08 fold, p < 0.001), phenylalanine (1.41 ± 0.02 fold, p < 0.001), tyrosine (1.24 ± 0.12 fold, p < 0.05), tryptophan (1.19 ± 0.07 fold, p < 0.05), glutamate (1.91 ± 0.05, p < 0.001), cysteine (1.52 ± 0.11, p < 0.01), threonine (1.71 ± 0.07 fold, p < 0.001) and proline (1.54 ± 0.01 fold, p < 0.001) were all increased by treatment with AAE.
Probably spared from being used as substrate for mitochondrial activity amino acids are likely stored intracellularly for other metabolic reactions (like protein production). In support of this hypothesis, taurine (1.52 ± 0.16 fold, p < 0.01) (a derivative of cysteine) and creatine, a derivative of arginine (1.60 ± 0.09 fold, p < 0.01), are both increase by AAE.
Mitochondrial and peroxisomal β-oxidation both represent an alternative fuel for hepatic mitochondria. In AAE treated HuH7 cells, we measured a significant decrease in the intracellular level of short chain acyl-carnitines (scfa-carnitines), suggestive of their utilization in the Krebs Cycle. These are produced by peroxisomes via β-oxidation of long chain FA and coupled to carnitine in order to be transported into the mitochondrial matrix and enter the TCA cycle. AAE treatment decreases the intracellular levels of butyril-carnitine (0.56 ± 0.02 fold, p
< 0.001), propionyl-carnitine (0.54 ± 0.05 fold, p
< 0.001) and valeryl-carnitine (0.50 ± 0.03 fold, p
< 0.001) (Figure 2
c) all terminal products of peroxisomal FAs catabolism and precursor of TCA cycle intermediate succinate.
Overall our metabolite profiling revealed that, in HuH7 cells, AAE stimulates glycolysis and β-oxidation, ultimately increasing mitochondrial respiration (Figure 3
and Table 1
). The substrate of β-oxidation seems to be represented by FAs either taken up from the extracellular medium or released by lipolysis from internal stores. Stimulation of membrane lipids hydrolysis by AAE seems to be further confirmed by the increased levels of alpha glyceryl phosphoryl choline (α-GPC; 1.45 ± 0.04 fold, p
value < 0.01) a byproduct of phospholipase activity. On the contrary, several anabolic reactions occuring in the cytosol (glycogenolysis, lactic fermentation, GSH synthesis) as well as anabolic reactions occurring in the Endoplasmic Reticulum (FA synthesis and cholesterogenesis) were all diminished in vitro by the treatment with AAE.
Compared to statins, inhibition of de-novo synthesis of cholesterol by AAE is the result of a different mechanism. Statins block selectively HMG-CoA reductase, inhibiting conversion of HMG-CoA into mevalonate, while AAE, on the contrary, modulates the entire metabolic choices of HuH7 halting the usage of citrate for lipogenesis and cholesterogenesis.