1. Introduction
Pork and pork products are highly consumed foods in developed countries [
1]. Even though they are a great source of protein, vitamins, and minerals, there is significant epidemiological evidence that correlates meat consumption with degenerative disease, like cardiovascular disease (CVD) [
1] or an imbalance in lipid profile. Therefore, there has been growing interest in the research field that evaluates quantitative and qualitative modifications to obtain functional meats and meat products [
2,
3]. Restructuring pork (RP), by partially adding or changing some of the meat components, would permit to incorporate active ingredients with potential functional effects [
2,
3]. Our research group has previously studied the effect of walnut-enriched meat products consumption on the antioxidant and lipoprotein profile of volunteers at CVD risk [
2]. In addition, the consumption of omega-3-enriched-reduced fat meat products partially reduced thrombogenesis, coagulation, and insulin-resistance markers [
4]. Moreover, we have evaluated the effect of seaweed-enriched-RP consumption on different CVD risk markers in cholesterol-fed rats. Results largely depended on the type and composition of seaweed included [
5].
It has been recognized that adjustments in the quality of dietary lipids, such as omega-3 polyunsaturated fatty acids (ω-3 PUFA), are important in the prevention of metabolic disorders [
6]. In this regard, interest in α-linolenic acid (ALA; 18:3 ω-3) as a functional ingredient has grown in recent years because of its association with improvements in plasma lipid concentrations and CVD [
7]. Nevertheless, there are few foods that contain adequate amounts to provide the benefits associated with ALA consumption [
8].
The seeds of
Salvia hispanica L., known as chia seeds, are one of the richest sources of ALA found in nature [
7,
8]. The lipid content varies from 60–80% of total lipids comprised of ALA and 40–20% of linoleic acid (ω-6). Rodent studies have proved that the intake of Chia oil may lower serum cholesterol, low-density lipoproteins (LDL), and triglycerides, and increase high-density lipoproteins (HDL) [
9]. Furthermore, other studies have suggested an improvement in adiposity, blood lipids, and insulin resistance in dyslipidemic rats after
Salvia hispanica treatment [
10]. The mechanism of this plasma lipid improvement for dietary chia is not completely understood. It is possible that ALA, as an eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids precursor, could play an important role in fatty acid metabolism in the liver, such as lipogenesis and fatty acid oxidation [
11].
Hydroxytyrosol (HxT) is a polyphenol that has been used as a functional ingredient, especially in PUFA-enriched meats, because of its powerful antioxidant capacity [
12], and in pre-cooked meat products enriched with ω-3 PUFA [
13]. Its antioxidant properties in experimental animals, when incorporated into RP in high-fat diets, have already been evaluated by our group [
14]. Additionally, it has been found that a reduction of triglycerides and LDL-cholesterol, and an increase of HDL-cholesterol levels, have been reported in diabetic rats [
15]. Cao et al. demonstrated the protective effect of supplementing diet with HxT in a high-fat-diet animal model that induced obesity, hyperglycemia, hyperlipidemia, and insulin resistance. This study proved the ability of HxT to decrease lipid accumulation [
16]. Although much information about the impact of omega-3 PUFA on lipids is available, their effects on lipoprotein number and composition have been scarcely reported [
17]. High-energy, saturated fat, and cholesterol diets have been proven to induce nonalcoholic steatohepatitis in aged rats [
14,
18]. A previous study of our group with Chia oil and HxT supplementation has demonstrated the antioxidant and anti-inflammatory effects of these ingredients [
19]. The results showed the protective effect of the experimental diets that reduced the oxidative damage through the regulation of glutathione levels by increasing glutathione reductase and decreasing glutathione peroxidase levels. Also, Chia oil supplementation, despite its high PUFA content, was able to reduce lipid peroxidation. Additionally, the findings suggested that Chia oil and HxT were exogenous stimuli that promoted the activation of the Nrf2 pathway to control the pro-oxidative response to dietary cholesterol and ageing [
19]. However, to the best of our knowledge few papers have tested how the inclusion of HxT or Chia to this animal model of nonalcoholic steatohepatitis affects the lipoprotein content and composition.
This study hypothesizes that Chia oil or HxT incorporated into a RP partially block the changes on lipoprotein profile induced by a high cholesterol/high saturated fat diet. In addition, the aim of this investigation was to determine the effects of the different experimental diets on lipoprotein profile and composition, SREBP-1c protein, and LDL receptor (Ldlr) gene expressions in aged rats.
4. Discussion
Quantitative and qualitative modifications of meat composition are emerging topics addressed to decrease the potential negative effects of a high meat products consumption on CVD [
2,
3]. The major aim of this study was to characterize the potential benefits of consuming Chia oil- or HxT-enriched pork on lipemia, lipoproteinemia, and lipoprotein profile of aged rats fed high-fat, high-energy, cholesterol-enriched diet. The latter diet has been reported to induce nonalcoholic steatohepatitis in aged rats [
14,
18]. Chia oil or HxT partially reversed alterations induced by cholesterol feeding, through normalizing VLDL total lipids and reducing VLDL proteins, as well as by modifying SREBP-1c and
Ldlr expressions, which are two important markers of lipid metabolism.
Based on the evidence of the present and previous works, we propose a probable mechanism that explains, on one hand, the effects of adding cholesterol to a high fat/high energy RP-diet and, on the other hand, the effects of including Chia oil and HxT in those RP-diets (
Figure 3).
The C rats demonstrated a lower dietary intake than that of growing rats that received seaweed-RP diets [
5]. However, results were similar to those observed by Nesic et al. [
32], which can be partially related to the ageing anorexic effect [
33]. There were no significant differences between feed intake and body weight in the HC group, in comparison with results for younger cholesterol-fed rats [
5,
22,
29]. Thus, although body weight gain was significantly reduced in all rat groups fed the experimental cholesterol-enriched diets, aging may alter the effect of cholesterol intake on body weight. Nevertheless, the findings could also be a result of the high-energy/high-saturated fat content of experimental diets. As in cholesterol-fed animals [
34], HC rats registered higher fecal excretion than C rats (
Figure 3, point 1), suggesting a reduction in diet digestibility of the HC animals that would contribute to both a lower body weight gain and adipose tissue mass. A significant negative correlation was found between body weight gain and fecal fat deposition (
p < 0.01), which supports previous explanations and could explain the decrease of final body weight in the cholesterol fed animals (
Figure 3, point 2). Our group has reported that the dietary cholesterol supplementation in rats decreased the adipose tissue stores by increasing the hormone sensitive lipase (HSL) expression, a mechanism linked to plasma cholesterol regulation [
35].
In agreement with the aging effects observed in non-cholesterol fed rats [
36], C rats registered moderately higher plasma cholesterol and phospholipids levels than younger rats [
5,
28,
34]. In fact, 37.5% of C animals presented cholesterolemia values higher than 100 mg/dL, cut-off point of rat hypercholesterolemia [
37]. Dietary cholesterol supplementation increased total lipids and cholesterol levels (19% and 32%, respectively). In addition, contingence test showed significant differences (
p < 0.05) of distribution between hyper- and normocholesterolemic rats in HC and C groups, since all the HC rats were moderately hypercholesterolemic (>2.52 mmol/L).
Nonetheless, in comparison with previous studies carried out by our group in younger animals [
5,
24], the 0.8 mmol/L cholesterol increase due to dietary cholesterol + cholic acid was smaller than expected. Erdinçler et al. [
36] also observed a small but substantial increase in plasma cholesterol in aged rats fed similar quantities of cholesterol. At a certain level, CHIA blocked the hyperlipidemic effect of cholesterol shown by HC group by means of lowering cholesterolemia, triglyceridemia, phospholipemia, total lipids, and FFA, by increasing fatty acid β-oxidation (
Figure 3, point 3) [
25]. These results are consistent with those of previous studies where chia administration, as a supplement of fiber and fat, reduced lipid parameters in rats [
38].
With respect to HxT, the hypotriglyceridemic and hypophospholipemic mechanism could be related to the thermogenic properties of HxT, as proposed in previous studies [
19,
39]. The induced thermogenesis would decrease the lipid accumulation and increase the fat mobilization from adipose tissue, giving rise to a lower plasma FFA amount in these rats (
Figure 3, point 4).
The antioxidant defense system improvement by CHIA and HxT, previously reported by our group, could be partially related to the hypolipemic effects found in the present paper, as it plays an important role in the reduction of lipid accumulation in the liver [
19]. This information is relevant, as it is well known that fatty liver disease can be highly prevented through the antioxidant defense system activation [
19].
C rats displayed a characteristic rat lipoprotein profile with low LDL and high HDL levels [
5,
27,
34,
37]. Rat has been categorized as HDL-animal [
26,
37], exhibiting a very effective uptake of VLDL through the
Ldlr and a lesser apolipoprotein (apo) B transfer from VLDL to LDL. Nonetheless, it has been reported that age induces a lower
Ldlr expression [
40], which would in part explain the contribution of the IDL + LDL fraction to total plasma lipids in C rats.
HC rats showed increased VLDL fraction levels, likely due to their low Ldlr gene expression and high SREBP-1c protein expression. It has been reported that an elevation on SREBP-1c, expression occurs as a consequence of a reduction in the pool of hepatic free cholesterol, which in turn would depend on the availability of FFA in the liver [
41]. Viejo et al. [
42] found a marked increase in both hepatic cholesterol esters and the esterified/free cholesterol ratio in rats fed high-cholesterol diets. These authors suggested that this esterification would occur as a mechanism to maintain reduced levels of free cholesterol, thus attempting to maintain reduced levels of
Ldlr. These considerations complied with the observed increase of SREBP-1c and FFA in the HC group (
Figure 3, point 5).
A relevant mechanism linked to the hyperlipidemic effects of HC diet is explained through the high plasma FFA levels in those animals. FFA are one of the most powerful signals to induce liver triglyceride and other lipid synthesis, which are also linked to the decrease in adipose tissue weight [
35] (
Figure 3, point 6). Significant correlations between plasma lipids and VLDL total mass with FFA were found in the present paper.
Lipoprotein levels and composition suggest that there was a reasonably higher number of cholesterol-enriched VLDL in HC rats than in their C equivalents (
Figure 3, point 7). The IDL + LDL fraction was also richer in cholesterol and poorer in triglycerides. In addition, the protein content of this fraction was higher, proposing that there was an elevated number of these particles in plasma. Vázquez-Velasco et al. [
43] found that IDL + LDL increased in cholesterol-fed fa/fa rats. The results of our study agree with previous publications that reported the presence of cholesterol enriched-VLDL (β-VLDL) [
28,
44]. These β-VLDL have been defined as atherogenic lipoproteins for the rat [
45]. The reduction of HDL-total mass in HC rats was similar to previous results in hypercholesterolemic rats [
5,
43], probably linked to HDL uptake by the scavenger receptor B-1 (SRB1) to increase cholesterol excretion via bile [
46]. Since the HDL composition was unaffected but their total mass concentration was diminished, it can be accepted that there was a reduction in the number of HDL particles.
As in HC rats, the VLDL fraction in CHIA or HxT rats was cholesterol-enriched. However, both groups showed considerably minor levels of protein and total mass in the VLDL fraction, suggesting a clear reduction in the number of VLDL particles. This mechanism has not been completely understood, but it seems to involve a fall in the triglycerides and protein amounts available for the VLDL synthesis, indicative of the hypotriglyceridemic mechanism proposed for ω-3 fatty acids [
25]. The high concentration of ω-3 fatty acids in Chia oil has been found to decrease VLDL levels [
47]. It can be hypothesized that CHIA would reduce phospholipids, triglycerides, and therefore VLDL particles through ω-3 fatty acid β-oxidation [
25] and by increasing the pool of fatty acids for liver cholesterol esterification [
44], while HxT would increase the thermogenic process. CHIA or HxT did not increase
Ldlr expression vs. HC rats, although it tends to increase by 33% and 22% for CHIA and HxT, respectively. This may be as a result of the capacity of CHIA or HxT to induce relocation of lipids in the body exerting a hepatoprotective effect. This is achieved by decreasing lipid accumulation in the liver and in visceral adipose tissue. In addition, VLDL synthesis is reduced making the increased expression of the SREBP-1c—a key marker in the lipogenesis pathway—unnecessary [
48] (
Figure 3, point 8).