1. Introduction
Phosphatidylglycerol (PG) is a naturally occurring phospholipid (PL) with glycerol as a negatively charged polar head group. PG is found in bacteria as a major PL; in plants and mammals, PG is also detected as a minor PL with a varied fatty acid composition [
1]. Furthermore, in several species of microalgae,
n-3 polyunsaturated fatty acid (PUFA) binding PG (
n-3 PUFA-PG) was contained, and was even found to be a major lipid component [
2,
3].
n-3 PUFAs are well known to have health benefits [
4]. We usually intake
n-3 PUFAs through the consumption of fish or fish oil to maintain adequate amounts of
n-3 PUFAs in the body. Interestingly, the chemical forms of the lipids binding
n-3 PUFAs have been suggested to affect the health benefit and bioavailability of
n-3 PUFAs. Shirouchi et al. [
5] described the superiority of
n-3 PUFA-PC in prevention or alleviation obesity-related disorders through the suppression of fatty acid synthesis, enhancement of fatty acid beta-oxidation compared egg PC in Otsuka Long-Evans Tokushima fatty rats. Numerous studies have evaluated the bioavailability of several lipid forms, such as triacylglycerol (TAG), PL, ethyl esters, and monoacylglycerol (MAG) (mainly binding eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)). DHA binding PL (DHA-PL) and DHA binding MAG (DHA-MAG) are suggested to be efficient carriers of dietary DHA in erythrocytes and plasma lipids when compared to DHA binding TAG (DHA-TAG) [
6]. Liu et al. determined a higher efficacy of dietary DHA provided as PL than as TAG for brain DHA accretion in neonatal piglets [
7]. Another prior study demonstrated that compared to fish oil TAG, roe-derived PL (
n-3 PUFA containing PL (
n-3 PUFA-PL)) administration enhanced lymphatic T DHA-PL absorption in unanesthetized rats, and plasma
n-3 PUFAs are important as the supplying pool of
n-3 PUFAs into various tissues [
8]. However, in another study of mouse fed DHA-PL and DHA-TAG, DHA accretion in tissues did not differ between the administration of purified PL and TAG forms [
9].
PG is a functional lipid with excellent emulsifiability and is expected to be applied in functional foods, cosmetics, and drug delivery. A previous study reported that palmitoyl-oleoyl-PG inhibits eicosanoid production in macrophages stimulated by
Mycoplasma pneumoniae, insofar as saturated PG and saturated and unsaturated phosphatidylcholines did not have a significant effect on
M. pneumoniae-induced arachidonic acid (ARA) release [
10]. It has been demonstrated as well that PG-liposome decreases TNF-α production of lipopolysaccharide-stimulated macrophages in vitro [
11]. On the other hand, we reported the preparation of
n-3 PUFA-PG from salmon roe PL through phospholipase D (PLD)-mediated transphosphatidylation [
12,
13].
n-3 PUFA-PG has been given attention as a highly functional lipid with properties of both PG and
n-3 PUFAs. We found that EPA and DHA at the s
n-2 position of
n-3 PUFA-PG were rapidly liberated by the pancreatic phospholipase A
2 in vitro digestion model. However, there is little information about the beneficial health functions of dietary
n-3 PUFA-PG and the incorporation and tissue accumulation of PUFAs from
n-3 PUFA-PG. In the present study, we examined the effects of
n-3 PUFA-PG on white adipose tissue (WAT) weight, blood glucose level, and serum and liver lipids of diabetic/obese KK-
Ay mice to explore its anti-obesity and anti-diabetic effects. Further,
n-3 PUFA accretion in the tissues of KK-
Ay mice from dietary
n-3 PUFA-PG was analyzed compared to
n-3 PUFA-TAG and SoyPC diets.
4. Discussion
The chemical structure of lipids has been reported to influence
n-3 PUFA bioavailability [
1]. It is well-documented that the
n-3 PUFA-TAG is hydrolyzed into free fatty acids and s
n-2-MAG by pancreatic lipase in the intestinal lumen [
17]. After absorption of these hydrolysates, PL or TAG is re-synthesized in the small intestine epithelium and migrates into the lymph. On the other hand, PC is hydrolyzed into 1-acyl-lyso-PC and free fatty acids by pancreatic phospholipase A
2 in the intestine [
18]. A higher bioavailability of EPA and DHA from dietary
n-3 PUFA-PL compared to
n-3 PUFA-TAG has been reported using krill oil (EPA and DHA are mainly bound to PL) [
8]. The previous research demonstrated, with high levels of confidence, that krill oil is effective for the management of hyperlipidemia at lower and equal doses and is significantly more effective than fish oil for the reduction of glucose, triglycerides, and LDL levels [
19]. Another study indicates that krill oil is more effective than fish oil in increasing
n-3 PUFA, thus reducing the
n-6/
n-3 PUFA ratio [
20]. Krill oil with higher PL levels enhanced the bioavailability of
n-3 PUFA compared to krill oil with lower PL levels [
21]. Thus, the bioavailability of
n-3 PUFAs has a close relationship with their esterified lipid forms.
In marine fish roe,
n-3 PUFAs are mainly bound at the
sn-2 position of PL [
22]. Previously, we reported
n-3 PUFA-PG preparation from salmon roe PL through PLD-mediated transphosphatidylation, and
n-3 PUFAs binding to PG were predominately distributed at the s
n-2 position of the glycerol backbone. PG is an essential biological component with important roles, such as cellular functions, in all eukaryotes and some prokaryotes [
23]. In mammals, PG is a minor PL component of many intracellular membranes [
24]. In keratinocytes, PG species containing PUFAs were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with MUFAs were effective at promoting proliferation in slowly dividing ones [
25]. However, the health benefits of dietary
n-3 PUFA-PG remain largely unknown. In addition, there is no information about the bioavailability of
n-3 PUFAs from
n-3 PUFA-PG after its digestion and absorption in body. For the utilization of
n-3 PUFA-PG in functional foods and nutraceuticals, information regarding the health benefits and bioavailability of
n-3 PUFAs released from
n-3 PUFA-PG is important. Since obesity is recognized as worldwide problem because of risk factor for diabetes, hypertension and dyslipidemia to develop metabolic syndrome, we therefore examined the effect of
n-3 PUFA-PG on WAT weight and blood glucose level, as well as the serum and liver lipid levels of type 2 diabetic/obesity model KK-
Ay mice to explore its preventive and alleviative activities. Further, the tissue accumulation of
n-3 PUFAs was also analyzed for the first time in KK-
Ay mice by the supplementation of the
n-3 PUFA-PG diet compared with an
n-3 PUFA-TAG diet.
In this study, 2% n-3 PUFA-PG or n-PUFA-TAG with approximately equal amounts of EPA and DHA in the diets did not affect the body and WAT weight gain or the blood glucose level of obese/diabetic KK-Ay mice compared to SoyPC after longitudinal (over 30 days) experimental feeding. To examine the anti-obesity effect of n-3 PUFA-PG, long-term feeding or more n-3 PUFA-PG in the diet might be required. On the other hand, 2% n-3 PUFA-PG in the diet significantly decreased the serum total cholesterol and non-HDL cholesterol as much as the n-3 PUFA-TAG. It is noteworthy that the n-3 PUFA-PG better alleviated lipid accumulation in the liver than SoyPC. The n-3 PUFA-TAG did not significantly reduce hepatic TL and NL compared to SoyPC, although no significant difference was found in the TL, NL, and PL between the n-3 PUFA-PG and n-3 PUFA-TAG groups. These data indicate that n-3 PUFA-PG exhibits serum cholesterol and hepatic lipid reduction in diabetic/obese KK-Ay mice. The decreasing effect by n-3 PUFA-PG is suggested to have an identical or superior effect to the n-3 PUFA-TAG, and could be used as n-3 PUFAs source to improve the diabetic/obese associated lipid metabolism. To clarify the preventive effect of n-3 PUFA-PG on metabolic syndrome-related parameters, it is required for further investigation by using diet-induce obesity and diabetic mice and normal mice.
We further analyzed
n-3 PUFA accumulation in several tissues of KK-
Ay mice fed an
n-3-PUFA-PG diet. In the small intestine, liver and perirenal WAT, dietary
n-3 PUFA-PG significantly elevated EPA, DPA
n-3, and DHA, and reduced ARA at the same level as the
n-3 PUFA-TAG diet. The
n-3 PUFA-PG is a highly applicable lipid because of its excellent emulsifiability. In addition, several algal lipids, which have gained wide interest in various application in nutraceuticals, also contained
n-3 PUFA-PG [
26]. Therefore, the present study shows important results that
n-3 PUFA-PG is an available dietary lipid source to supply
n-3 PUFAs in the body.
DHA is one of the most abundant fatty acids in the brain to regulate important physiological functions [
27]. The synthesis rate of DHA from ALA and EPA is very slow in an animal’s body. Therefore, the uptake of dietary DHA is necessary to maintain the essential levels [
28]. Tracer studies indicate that phospholipid DHA targets the brain more effectively than DHA-TAG, although how this translates into higher brain DHA concentrations is not clearly understood [
29]. Another study found that DHA esterified to PC, PE, or phosphatidylserine was more efficient at targeting the brain than DHA esterified to TAG in cortex and serum lipids [
30]. In the present study,
n-3 PUFA-PG increased DHA in the brain of diabetic/obese KK-
Ay mice. Interestingly, DHA accreted in the brain by
n-3 PUFA-PG, but not by
n-3 PUFA-TAG, was significantly higher than that of SoyPC. However, there was no significant difference between the
n-3 PUFA-PG and
n-3 PUFA-TAG groups, and the DHA content in the
n-3 PUFA-PG diet was slightly higher than that in the
n-3 PUFA-TAG diet. To clarify the
n-3 PUFA-PG property needed to transport DHA in the brain, further investigation following a long feeding period and higher dose feeding is required.