Involvement of nutrients and nutritional mediators in mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene expression

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (HMGCS2) catalyses the first step of ketogenesis and is critical in various metabolic conditions. Several nutrient molecules were able to differentially modulate HMGCS2 expression levels. Docosahexaenoic acid (DHA, C22:6, n-3), eicosapentaenoic acid (EPA, C20:5, n-3), arachidonic acid (AA, C20:4, n-6), and glucose increased HMGCS2 mRNA and protein levels in HepG2 hepatoma cells, while fructose decreased them. The effect of n-6 AA resulted significantly higher than that of n-3 PUFA, but when combined all these molecules were far less efficient. Insulin reduced HMGCS2 mRNA and protein levels in HepG2 cells, even when treated with PUFA and monosaccharides. Several nuclear receptors and transcription factors are involved in HMGCS2 expression regulation. While peroxysome proliferator activated receptor α (PPAR-α) agonist WY14643 increased HMGCS2 expression, this treatment was unable to affect PUFA-mediated regulation of HMGCS2 expression. Forkhead box O1 (FoxO1) inhibitor AS1842856 reduced HMGCS2 expression and suppressed induction promoted by fatty acids. Cells treatment with liver X receptor alpha (LXRα) agonist T0901317 reduced HMGCS2 mRNA, indicating a role for this transcription factor as suppressor of HMGCS2 gene. Previous observations already indicated HMGCS2 expression as possible nutrition status reference: our results show that several nutrients as well as specific nutritional related hormonal conditions are able to affect significantly HMGCS2 gene expression, indicating a relevant role for PUFA, which are mostly derived from nutritional intake. These insights into mechanisms of its regulation, specifically through nutrients commonly associated with disease risk, indicate HMGCS2 expression as possible reference marker of metabolic and nutritional status.

KEYWORDS : fructose, gene expression, glucose, HMGCS2, insulin, PUFA, transcription factors

1 | INTRODUCTION

Endogenous metabolic pathways and exogenous factors, including nutrient molecules originating from diet, affect most cellular processes, considerably influencing human health state and susceptibility to diseases development. In particular, dietary intake, besides reintegrat- ing metabolic balance, provides bioactive molecules that are selec- tively able to modulate molecular mechanisms involved in the etiology of numerous pathological conditions, especially cardiovascular and neoplastic diseases (Alissa & Ferns, 2017; Maehre, Jensen, Elvevoll, & Eilertsen, 2015; Song, Garrett, & Chan, 2015). Numerous bioactive nutrients are being progressively identified and their chemopreventive effects are being described at clinical and molecular mechanism levels, revealing the ability of some of them to regulate gene expression. Several macro (amino acids, carbohydrates, fatty acids, and sterols) and micro (minerals, vitamins) nutrients were found able to control the expression of genes directly involved (or not) in their own metabolism (Bruhat et al., 2009; Deckelbaum, Worgall, & Seo, 2006; Joven et al., 2014; Minieri & Di Nardo, 2007; Pégorier, Le May, & Girard, 2004). Hormonal regulation is considered a major mediator of gene expression control in response to nutrients variations. The discovery and characterization of several transcription factors, mediating nutrients responses, allowed to demonstrate that they can also directly affect nutritional related gene expression (Meugnier, Rome, & Vidal, 2007).

Many studies reported that several transcription factors are directly and/or indirectly modulated by polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, C22:6, n-3), eicosapen- taenoic acid (EPA, C20:5, n-3), and arachidonic acid (AA, C20:4, n-6). In fact, PUFA can affect several metabolic pathways through two mechanisms: (1) the direct binding to some nuclear receptors, such as peroxisome proliferator activated receptor (PPAR), liver X receptor (LXR), and hepatic nuclear factor-4α (HNF-4α) and (2) the indirect regulation of the nuclear amount of some transcription factors, such as sterol regulatory element binding protein-1 (SREBP-1), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), carbohydrate regulatory element binding protein (ChREBP), and maxlike factor X (MLX) (Duplus & Forest, 2002; Jump, 2008; Marion-Letellier, Savoye, & Ghosh, 2015). Mechanisms of gene expression regulation mediated by carbohydrates are less characterized. Insulin is always described as the main mediator of glucose effects at transcriptional level. However, several studies suggested that also carbohydrates, like other key nutrients, can directly control gene expression (Mirasierra & Vallejo, 2016; Patel et al., 2015; Won, Rhee, & Ko, 2009). Glucose induced L-type pyruvate kinase (LPK) gene expression, regardless of insulin, in cultured hepatocytes (Doiron, Cuif, Kahn, & Diaz-Guerra, 1994). ChREBP transcription factor seems a key player of carbohydrate- induced transcriptional activity in liver (Jois & Sleeman, 2017; Uyeda, Yamashita, & Kawaguchi, 2002), but detailed mechanisms of this regulation are still to be fully described.

Human mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) catalyzes the first step of ketogenesis, the condensation of acetoacetyl-CoA and acetyl-CoA to form HMG-CoA (Hu et al., 2017). Ketone bodies (β-hydroxybutyrate, acetoacetate, and acetone), which are later produced, are essential products allowing the transfer of energy from liver to extra hepatic tissues like brain, heart, and kidneys (Grabacka, Pierzchalska, Dean, & Reiss, 2016; Robinson & Williamson, 1980; Williamson, Bates, & Krebs, 1968), especially during fat feeding, fasting, and other conditions in which carbohydrate availability is limited (Hegardt, 1999). In these conditions, both ketogenesis and HMGCS2 expression are stimulated. HMGCS2 expression can be regulated both at transcriptional and posttransla- tional level (Boukaftane & Mitchell, 1997; Cook, Lavrentyev, Pham, & Park, 2017; Shimazu et al., 2010). We previously demonstrated that HMGCS2 expression was regulated by diet derived nutrients and by individual polyunsaturated fatty acids (De Rosa et al., 2015). HMGCS2 mRNA resulted increased in HepG2 cells treated with sera from hypercholesterolemic patients when compared to cells treated with sera from normocholesterolemic subjects, indicating a relevant effect of the nutrition status as typical cause of dyslipidemia.

Transcription of HMGCS2 gene is regulated through the binding of the PPAR-retinoid X receptor (RXR) complex to a nuclear receptor- responsive element (NRRE) on the promoter (Rodríguez, Gil-Gómez, Hegardt, & Haro, 1994; Rodríguez, Ortiz, Hegardt, & Haro, 1998). Furthermore, other specific sequences on HMGCS2 promoter allow regulation by transcription factors of Fox family, with an induction that is suppressed by insulin (Nadal, Marrero, & Haro, 2002). Other nuclear receptors able to affect HMGCS2 expression are COUP transcription factor (COUP-TF) and HNF-4, functioning mostly as transcriptional inhibitors (Rodríguez et al., 1998).

In the present study, we analyzed the ability of nutrients such as DHA, EPA, AA, glucose, and fructose to regulate HMGCS2 gene expression and the relationship of these effects with insulin action. We also evaluated the role of several transcription factors and the effect of several nutrient combinations.

2 | MATERIALS AND METHODS

2.1 | Reagents

Fatty acids, glucose, fructose, insulin, bovine serum albumin fatty acid free, WY14643, T0901317, and all other chemicals were purchased from Sigma–Aldrich (St. Louis, MO). AS1842856 was from Calbiochem Merck Millipore (Darmstadt, Germany). Cell culture reagents were purchased from Euroclone S.p.A. (Pero, Italy). Nitrocellulose, hyperfilms and ECL Western blot system were provided by GE Healthcare Europe GmbH (Milan, Italy). Protein assay reagent and protein molecular weight standards were from Bio-Rad Laboratories (Hercules, CA). Antibody versus HMGCS2 (ab137043) were from Abcam (Cambridge, UK); antiboby against GAPDH and normal rabbit IgG were from Santa Cruz Biotechnologies (Heidelberg, Germany). DNase I and SYBR Green I Master-Mix were from Roche Applied Science (Mannheim, Germany). Trizol Reagent and SuperScript® II Reverse Transcriptase kit were from Invitrogen Co. (Carlsbad, CA). Primers were custom synthesized by Primm (Milan, Italy).

2.2 | Cell cultures and treatments

Human hepatoma HepG2 cell line (obtained from American Type Culture Collection, Manassas, VA) was cultured in Eagle’s minimum essential medium supplemented with 10% (v/v) fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. For experiments, cells were seeded at a density of 7 × 105 per 60-mm dish and allowed to adhere for 24 hr. Medium was then replaced with fresh serum-free medium with specific substances of each experiment. Fatty acids (DHA, EPA, AA 25, 50, 100 µM) were dissolved in ethanol and each fatty acid was mixed with fatty acid-free bovine serum albumin at 2:1 molar ratios before addition to medium. WY14643, AS1842856, and T0901317 were dissolved in dimethyl sulfoxide (DMSO) and used at 50, 1, and 5 µM respectively, for 24 hr. Glucose and fructose were dissolved in PBS and used at 25 mM for 24 hr. Used concentrations of PUFA, glucose, fructose, insulin, WY14643, AS1842856, and T0901317 were selected on the basis of preliminary experiments and previous literature experiences to obtain the highest effects. Control samples were made culturing cells with vehicles used for each substance.

2.3 | RNA extraction and reverse transcription

Total RNA was isolated from treated cells as previously described (Caputo, Zirpoli, et al., 2014). Approximately 3 µg of RNA were reverse transcribed in a total volume of 20 µl RT buffer with 20 U SuperScript® II Reverse Transcriptase, 0.5 mg/ml random primers, deoxynucleotide (dNTP) mix (each 10 mM), 0.1 M dithiothreitol. Thermal conditions for reverse transcription were 42°C for 50 min, 70°C for 15 min, and 37°C for 20 min. In the last step, RNAse H was added.

2.4 | Real-time PCR

Real-time PCR was performed with Light-Cycler® 480 (Roche Diagnostics GmbH, Mannheim, Germany) using SYBR Green detection in a total volume of 20 µl with 1 µl of forward and reverse primers (10 µM) and 10 µl of SYBR Green I Master-Mix (Roche Diagnostics GmbH, Mannheim, Germany). Reactions included an initial cycle at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 56°C for 5 s, extension at 72°C for 15 s Values were determined from standard curve generated from serial cDNA dilutions and normalized to 18S.

To analyze HMGCS2 expression level the following primer sets were used: forward human HMGCS2 5′-GCT TCT CCC CGT GAA TCA TA-3′; reverse human HMGCS2 5′-ACC ATA AGC CCA GGA CAG T-3′; forward human 18S 5′-CGA TGC TCT TAG CTG AGT GT-3′; reverse human 18S 5′-GGT CCA AGA ATT TCA CCT CT-3′. Differences between mean values were evaluated for statistical significance using the unpaired Student’s t-test. Each treatment was performed at least in three biological replicates.

2.5 | Cell protein extraction and Western blot analyses

After treatments, cells were washed and lysed as previously described (Caputo, De Rosa, et al., 2014). Thirty micrograms of total proteins from each extract were separated by 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes in a cooling system at 100 V for 1 hr. Membranes were saturated for 1 hr at room temperature with 0.1% Tween-20, 5% dry milk or BSA in PBS. Membranes were then incubated with antibody against human HMGCS2 diluted 1:1,000, overnight at 4°C, washed several times and incubated with peroxidase- conjugated secondary antibody (anti-rabbit diluted 1:5,000) for 1 hr at room temperature. Specific bands were then detected by ECL Western blot system. Antibody against glyceraldehyde 3-phosphate dehydroge- nase (GAPDH) was used as normalizing control. Densitometry of bands was performed with ImageJ software (http://rsbweb.nih.gov/ij/ download.html). Molecular sizes were evaluated referring to protein molecular weight standards. For these investigations each treatment was performed at least in biological triplicate.

2.6 | Statistical analysis

Data are presented as mean ± standard deviation. Differences between treatment groups were analyzed by Student’s t-test. Differ- ences were considered significant when p < 0.05.

3 | RESULTS

3.1 | PUFA, glucose, and fructose effects on HMGCS2 expression

We first examined the effects of several concentrations of DHA, EPA, and AA on HMGCS2 mRNA levels. DHA and EPA were selected as most relevant dietary n-3 fatty acids, while AA as relevant dietary n-6 PUFA. HepG2 cells were incubated for 24 hr with 25, 50, and 100 μM fatty acids and HMGCS2 mRNA was measured by real-time PCR. As shown in Figure 1a, considering 25 and 50 μM concentrations, all assayed fatty acids increased HMGCS2 mRNA in a dose-dependent manner. n-6 AA 50 μM enhanced most significantly HMGCS2 mRNA amount compared to n-3 DHA and EPA. Conversely, treatments with 100 μM fatty acids did not cause significant increases of HMGCS2 mRNA. This is essentially due to the fact that, at this concentration, HepG2 viability was strongly reduced (data not shown). Therefore, we used 50 μM fatty acids for all further experiments. Western blot analyses of HMGCS2 protein expression showed a similar modulation trend after 24 hr-incubations with 50 μM fatty acids (Figure 1b).

The effects of two other relevant nutrients, glucose and fructose, on HMGCS2 expression were also analyzed. In the same previous experimental conditions, we determined HMGCS2 mRNA and protein levels after incubations with each 25 mM monosaccharide. As reported in Figure 2a, the two monosaccharides oppositely affect HMGCS2 expression. In particular, glucose was able to up-regulate HMGCS2 mRNA while fructose down-regulated it. In this case too, Western blot investigations showed a similar modulation trend of HMGCS2 protein, after treatments with monosaccharides (Figure 2b).

3.2 | Effect of insulin on nutrient-mediated HMGCS2 modulations

It is known that insulin inhibits transcription of HMGCS2 (Nadal et al., 2002). Therefore, we evaluated if observed nutrient-mediated effects on HMGCS2 expression were affected in presence of insulin. Cells were treated with fatty acids or monosaccharides alone, or in combination with insulin. After 24 hr HMGCS2 mRNA and protein amounts were determined through real-time PCR and Western blot assays, respectively. We first verified 100 nM insulin ability to decrease HMGCS2 mRNA and protein levels in HepG2 cell line (Figure 3a). We then compared the effects of PUFA and mono- saccharides treatments in presence or in absence of insulin (Figure 3b–c). We found insulin able to repress PUFA induction of HMGCS2 mRNA expression to about one sixth of the value derived from the increase produced from each single fatty acid (0.166 for DHA, 0.158 for EPA, and 0.164 for AA). Reduction of protein levels, as densito- metrically determined, were also similar among the three fatty acids with a ratio of 0.393 for DHA, 0.401 for EPA, and 0.514 for AA, when comparing each value with and without insulin (Figure 3b). When glucose was administrated in combination with insulin, once again its stimulatory effect on HMGCS2 expression was eliminated and value reduced to about one third of the control (Figure 3c). However, an additive inhibitory effect was observed when insulin and fructose were simultaneously added (Figure 3c), as both of them individually produced a reduction of HMGCS2 mRNA and protein.

FIGURE 1 Up-regulation of HMGCS2 gene expression by DHA, EPA, and AA. (a) HepG2 cells were treated with 25, 50, and 100 µM PUFA and, after 24 hr, total mRNA was extracted. HMGCS2 mRNA levels were determined by Real-time PCR, calculating ratios between 18S normalized signals from treated and control cells. Reported data were expressed as means of three independent experiments ± standard deviations. **p < 0.01. (b) HMGCS2 protein levels were determined by Western blot analysis in cells treated with 50 µM PUFA for 24 hr. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as normalization reference.Images shown are representative of three independent experiments.

3.3 | Effects of PPAR-α agonist WY14643 on HMGCS2 gene expression

We previously reported that PPAR-α activation increased HMGCS2 expression (De Rosa et al., 2015). To deepen information on molecular mechanisms involved in this regulation, we incubated cells with combinations of each fatty acid or insulin and 50 μM WY14643 (WY), a synthetic agonist of PPAR-α (Bernardes et al., 2013) and then HMGCS2 mRNA levels were measured by real-time PCR. Graph in Figure 4a confirms that PPAR-α activation significantly increased HMGCS2 mRNA level while, at the same time, PPAR-α agonist was unable to affect PUFA-mediated regulation of HMGCS2 expression. Insulin and WY cotreatments did not affect HMGCS2 mRNA basal level, balancing insulin-mediated down-regulation, and PPARα- induced up-regulation effects (Figure 4b).

FIGURE 2 Modulation of HMGCS2 gene expression by glucose (GLU) and fructose (FRU). (a) HepG2 cells were treated with 25 mM glucose or fructose and, after 24 hr, total mRNA was extracted. HMGCS2 mRNA levels were determined by Real-time PCR, calculating ratios between 18S normalized signals from treated and control cells. Reported data were expressed as means of three independent experiments ± standard deviations. *p < 0.05,**p < 0.01. (b) HMGCS2 protein levels were determined by Western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as normalization reference. Images shown are representative of three independent experiments.

3.4 | Effects of FoxO1 inhibitor AS1842856 on HMGCS2 gene expression

In our previous work (De Rosa et al., 2015), treatments with synthetic FoxO1 inhibitor AS1842856 (AS), produced a significant down-regulation of HMGCS2 mRNA and protein levels, suggesting a role also for this transcription factor in HMGCS2 expression regulation. In this study, we treated HepG2 cells with individual PUFA or insulin in presence of the same Fox01 inhibitor (1 μM). Figure 5a shows that increases of HMGCS2 mRNA induced by DHA, EPA, and AA were abolished by FoxO1 inhibition which was prevalent with DHA and EPA, while AA induction was only partially blocked. Treatments with insulin and AS caused a further decrease of HMGCS2 mRNA summing their individual inhibition effects (Figure 5b).

FIGURE 3 Down-regulation of HMGCS2 gene expression by insulin (Ins). Both HMGCS2 mRNA and protein amounts were determined after 24 hr-treatments with 100 nM insulin alone (a) and in combination with fatty acids (b) or in combination with monosaccharides (c). HMGCS2 mRNA levels were determined by Real-time PCR, calculating ratios between 18S normalized signals from treated and control cells and reported data were expressed as means of three independent experiments ± standard deviations. **p < 0.01. HMGCS2 protein levels were determined by Western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as normalization reference. Images shown are representative of three independent experiments.

3.5 | LXR involvement in regulation of HMGCS2 mRNA expression

Our previous work provided evidence of a direct binding of polyunsaturated fatty acids DHA, EPA, and AA to LXRα, reducing its activation capability (Caputo, De Rosa, et al., 2014). To verify if HMGCS2 modulation mediated by PUFA involves also LXRα signaling, we incubated cells with two concentrations of T0901317 (T0), a synthetic nonsteroidal LXRα agonist, and then determined HMGCS2 mRNA. LXRα activation reduced HMGCS2 mRNA amounts in a dose- dependent manner (Figure 6a).

FIGURE 4 Stimulation of HMGCS2 gene expression by WY14643 (WY) PPARα agonist. HepG2 cells were treated with 50 µM WY alone and in combination with fatty acids (a) or with insulin (b). After 24 hr, total cell RNA was extracted and HMGCS2 mRNA level was determined by Real-time PCR, calculating ratios between 18S normalized signals from treated and control cells and reported data were expressed as means of three independent experiments ± standard deviations.

To evaluate if treatments with LXR agonist were able to affect HMGCS2 up-regulation induced by PUFA, cells were treated with individual fatty acids or in combination with the most effective concentration of the agonist (5 μM). Figure 6b shows that LXR activation opposes the up-regulation of HMGCS2 mRNA levels caused by DHA, EPA and AA, almost completely for the first two and partially for the last. Finally, in presence of insulin, the T0901317-mediated HMGCS2 reduction appeared attenuated, as if this hormone was able to interfere with LXRα activation (Figure 6c).

3.6 | Inhibitory effect of PUFA combinations on HMGCS2 mRNA expression

Nutrients do not act individually in vivo but, as in serum, in combination with a variety of other substances. Therefore, we also evaluated the effect of fatty acids combinations on HMGCS2 expression. Cells were treated with three different combinations of fatty acids (DHA + AA, EPA + AA, DHA + EPA) and HMGCS2 mRNA was determined by real-time PCR. For these treatments, each single PUFA was used at concentration of 25 μM to obtain a final fatty acids concentration of 50 μM. Figure 7 shows that PUFA combinations were weakly (EPA + AA) or not at all (DHA + AA, DHA + EPA) capable to affect transcription of HMGCS2 gene, when compared to treatments with individual fatty acids used at the same concentrations, resulting in a negative synergy.

FIGURE 5 Down-regulation of HMGCS2 gene expression by AS1842856 (AS) FoxO1 inhibitor. HepG2 cells were treated with 1 µM AS alone and in combination with fatty acids (a) or with insulin (b). After 24 hr total cell RNA was extracted and HMGCS2 mRNA level was determined by Real-time PCR, calculating ratios between 18S normalized signals from treated and control cells, and reported data were expressed as means of three independent experiments ± standard deviations.

4 | DISCUSSION

Health state as well as several pathological conditions are often associated with the amount and/or the type of specific nutrient molecules included in diet (Du & Fang, 2016; Maehre et al., 2015; Willett, 1994). Several bioactive nutrients are able to affect a large number of metabolic processes and cellular functions, including gene expression (Marion-Letellier et al., 2015; Won et al., 2009). Control of gene expression mediated by nutritional molecules affects metabolism similarly, although independently, to hormonal regulations and may require the activation of specific transcription factors (Wolfrum, Asilmaz, Luca, Friedman, & Stoffel, 2004). HMGCS2 is a key enzyme that constantly controls ketogenesis, depending on metabolic conditions (Hu et al., 2017; Vilà-Brau, De Sousa-Coelho, Mayordomo, Haro, & Marrero, 2011). It is one of the highest induced genes in liver during fasting (Cheon et al., 2005). The amount of HMGCS2 mRNA is rapidly changed in response to cellular cyclic AMP (cAMP), insulin, dexamethasone and is greatly increased by starvation, fat feeding, and diabetes (Casals et al., 1992; Helenius et al., 2015; Serra et al., 1993).

FIGURE 6 Down-regulation of HMGCS2 gene expression by LXR agonist T0901317 (T0). HepG2 cells were treated with T0 alone (a) and in combination with fatty acids (b) or with insulin (c). After 24 hr total cell RNA was extracted and HMGCS2 mRNA level was determined by Real-time PCR, calculating ratios between 18S normalized signals from treated and control cells, and reported data were expressed as means of three independent experiments ± standard deviations. **p < 0.01.

Moreover, the highest HMGCS2 expression is found in liver, which is, in fact, the main organ responsible of ketone bodies synthesis.In the present study, we demonstrated that 25 and 50 μM DHA, EPA and AA were able to increase HMGCS2 mRNA and protein in a dose-dependent manner. AA appeared most effective in HMGCS2 expression induction compared to DHA and EPA, revealing a differential stimulation ability between n-6 and n-3 fatty acids. When used at 100 μM concentration, the same fatty acids showed no significant increases of HMGCS2 mRNA, probably because these doses are too toxic for HepG2 cells. However, treatments with fatty acids combinations (DHA + AA, EPA + AA, and DHA + EPA), used at a overall 50 μM (25 μM each) final concentration, did not show the same stimulatory effect on HMGCS2 expression induced by individual PUFA. Only one combination (EPA + AA) induced a significant increase of HMGCS2 mRNA, although lower when compared to that induced by individual molecules used at the same concentrations, suggesting that each fatty acid acts on HMGCS2 expression through distinct mechanisms interfering each other.

FIGURE 7 Effect of PUFA combinations on HMGCS2 gene expression. HepG2 cells were treated with 25 µM PUFA alone or with PUFA combination (50 µM final concentration) and after 24 hr, total mRNA was extracted. HMGCS2 mRNA levels were determined by Real-time PCR, calculating ratios between 18S normalized signals from treated and control cells. Reported data were expressed as means of three independent experiments ± standard deviations.

Also high concentrations of two monosaccharides, that is, glucose and fructose, differently affected HMGCS2 expression. Glucose increased HMGCS2 mRNA and protein levels, while fructose significantly decreased both of them. It is known that the most important control point of glycolysis is enzymatic activity of phosphofructokinase. Fructose bypasses this control point and this could cause an uncontrolled accumulation of intermediate metabo- lites, such as acetyl-CoA and then of ketone bodies. Furthermore, fructose is known to stimulate fatty acids biosynthesis in liver (Carmona & Freedland, 1989). Therefore, fructose inhibition of HMGCS2 resulting from our data, could be an homeostatic mechanism to prevent excessive ketogenetic activity. More recently, Rebollo et al. (2014) found fructose able to inhibit liver fatty acid oxidation by reducing, both in rat and human liver cells, PPAR-α expression and activity, which is also known to affect HMGCS2 expression. This molecular mechanism could account for fructose-mediated HMGCS2 down-regulation. In physiological conditions, effects induced by high glucose concentrations are counteracted by insulin action. Therefore, we subsequently evaluated the effects of monosaccharides (as well as of PUFA) on HMGCS2 expression in the presence of insulin.

It is reported that insulin decreases HMGCS2 gene expression when injected three times/day in diabetic rats (Casals et al., 1992) and an insulin response sequence (IRS) in the 5′-flanking region of this gene, contributing to the transcription repression induced by insulin in HepG2 cells, was identified (Nadal et al., 2002). We found insulin able to reduce HMGCS2 mRNA and protein levels in HepG2 cells after 24 hr treatments. This is in agreement with insulin metabolic role, since in a fed state insulin stimulates glycolysis, while acetyl-coA is mainly consumed in the Krebs cycle. Furthermore, when we treated cells with insulin combined with individual nutrients, their effect on HMGCS2 expression was reduced. In particular, AA + insulin returned HMGCS2 mRNA and protein levels to values comparable to those of control samples. All other combinations not only cancelled the effect of DHA, EPA and glucose, but also produced an higher reduction of the expression of HMGCS2. These data suggest that the inhibitory effect of insulin on HMGCS2 expression tends to prevail on fatty acids and glucose action. Therefore, typical diabetic ketosis may be also related to a low insulin-mediated HMGCS2 inhibition, resulting in an over- expression of this enzyme (above all in presence of high fatty acids amounts). Insulin + fructose instead summed their inhibitory effects on HMGCS2 expression, possibly acting on distinct cellular pathways.

HMGCS2 was previously identified among genes responsive to elevated expression of PPAR-α (Hsu, Savas, Griffin, & Johnson, 2001). Our previous studies also provided evidence of PPAR-α and FoxO1 involvement in regulation of HMGCS2 expression (De Rosa et al., 2015). In agreement with these investigations, we found synthetic PPAR-α agonist WY14643 (pirinixic acid) and synthetic FoxO1 inhibitor AS1842856, respectively able to up-regulate and down- regulate HMGCS2 mRNA after 24h-treatments. However WY14643 was unable to affect PUFA effects on HMGCS2 mRNA levels, when administrated in combination (WY + DHA, WY + EPA, WY + AA). These data confirm that both WY14643 and analyzed fatty acids are able to induce HMGCS2 expression and suggest that PUFA may have more affinity for PPAR-α receptor than WY14643, also considering that they were used at the same concentration (50 μM). When administrated in presence of PUFA, FoxO1 inhibitor AS1842856 (AS) effect appears to be dominant. Since usually insulin controls a lot of cellular functions through FoxO1 inhibition (Eijkelenboom & Burgering, 2013; Wang, Zhou, & Graves, 2014), these results seem consistent with those obtained from fatty acids and insulin combinations and suggest that insulin effect on HMGCS2 expression prevails on those mediated by fatty acids. Furthermore, taken together these data suggest that signals sent through FoxO1 transcription factor prevail on those sent through PPAR-α nuclear receptor. However, insulin and WY combination almost did not affect HMGCS2 mRNA expression balancing insulin-mediated down- regulation and PPAR-α-induced up-regulation effects, suggesting that PPAR-α-independent mechanisms may be involved in HMGCS2 up-regulation induced by fatty acids. Finally, when administrated in combination, insulin and AS summed their inhibitory effect on HMGCS2 expression. This probably may depend on the use of two different FoxO1 inhibition mechanisms: insulin leads FoxO1 phosphorylation, preventing its translocation into the nucleus, while AS inhibits FoxO1 binding to its promoter (Nadal et al., 2002; Zou et al., 2014). These two mechanisms could then be affected without competition between them, independently both limiting HMGCS2 expression and resulting in the cumulative inhibition.

The present study firstly demonstrates the involvement of liver X receptor α (LXRα) on HMGCS2 expression regulation. Liver X receptors α and β belong to a family of nuclear receptors that controls the expression of many genes involved in the regulation of cholesterol and lipid homeostasis in different tissues (Hong & Tontonoz, 2014). LXRα is abundantly expressed in liver, intestine, kidney, spleen, and adipose tissue, whereas LXRβ is ubiquitously expressed at a lower level (Hong & Tontonoz, 2014). We found that two concentrations of the LXRα agonist T0901317 inhibit HMGCS2 expression; so this transcription factor appears to act as an HMGCS2 gene suppressor. Polyunsaturated fatty acids of the n-3 and n-6 families, such as eicosapentaenoic acid, docosahexaenoic acid, and arachidonic acid, are considered LXR antagonists and we previously demonstrated a direct inhibitory interaction of PUFA on LXRα (Caputo, De Rosa, et al., 2014). Now, we find that LXRα activation inhibits PUFA-mediated HMGCS2 induction, suggesting that PPAR-α activation is not sufficient to stimulate HMGCS2 expression, but LXRα inhibition is also required. In our previous work we also demonstrated that arachidonic acid binds LXRα more strongly than other fatty acids (DHA and EPA) (Caputo, De Rosa, et al., 2014). Arachidonic acid could then inhibit more strongly LXRα, explaining its higher induction of HMGCS2 expression compared to that produced by DHA and EPA.

Nutritional status is one of the main factors affecting human health and, according to its quality, may either induce or prevent pathological conditions. Biochemical markers could be useful to identify, possibly in an early phase, nutritional status conditions that could favor development of several diseases. HMGCS2 expression could be then used on this purpose since our data highlight that is specifically affected by nutrients which are known to be associated with health perspectives. Our results show that high doses of fatty acids (in particular of n-6 fatty acids) or glucose stimulate HMGCS2 mRNA and protein expression, mostly when administrated in absence of insulin. However, insulin absence or resistance are typical of diabetic state, which is commonly associated with ketosis and strictly related with nutrition state. We previously found a relevant up-regulation of HMGCS2 mRNA in HepG2 cells treated with sera from hypercholes- terolemic patients (De Rosa et al., 2015). We also recently obtained preliminary observations showing that rats receiving a diet containing only saturated fats had a much higher expression of liver HMGCS2 mRNA (data not shown). Since combined PUFA produced much lower increases of HMGCS2, when compared to individual administration of each molecule, this suggests that their real effect on this expression within complete organism is limited and more complex when acting together and with all other factors affecting this activity. This could also explain the fact that each individual PUFA increases HMGCS2 while these molecules are generally known as beneficial and preventive. Nevertheless, HMGCS2 expression strongly affects the PPAR-α mediated response, which is fundamental in lipid metabolism regulation (Vilà-Brau et al., 2011). Therefore, in addition to provide more details on HMGCS2 expression mechanisms, our data demon- strate that HMGCS2 gene expression increases when cell metabolic conditions are exposed to negative nutritional effects, supporting the hypothesis that liver expression of this gene is a reference marker of metabolic and nutritional status.