1. Introduction
Numerous epidemiological studies have indicated a correlation between elevated dietary fat intake and an increased body mass index (BMI) in humans [
1]. The standard American diet derives approximately 35–40% of its energy from fat [
2], which is often considered “unhealthy” as it surpasses the 30% of total energy from fat [
3]. Intriguingly, a rise in obesity prevalence in the United States has been linked with a decrease in fat and calorie consumption, a phenomenon referred to as the “American paradox” [
4]. A recent systematic review analyzing dietary trends in relation to the onset of non-communicable diseases identified an inverse relationship between animal fat intake and these conditions [
5]. However, in scientific research, diet-induced obesity models (DIO) are frequently employed to mimic the Western dietary pattern, even though the impact of a closely formulated Western diet for rodents, taking into account macro- and micronutrient components, showed quite different effects on weight gain and biomarkers of metabolic function in mice compared to a 45% fat DIO [
6]. DIO models often contain a notably high fat content, accounting for roughly 59% of energy from fat [
7,
8,
9,
10,
11], which is over 3.5 times the suggested 16.7% energy from fat for growing laboratory rats [
12]. Transposing these values to human equivalents, tripling dietary fat consumption would substantially surpass the tolerable fat intake limits for humans. Genetic overnutrition models, such as the
fa/
fa (leptin receptor) Zucker rats or the
db/
db and
ob/
ob (leptin) mice, present a more comparable representation of diet-induced human obesity conditions [
13,
14,
15]. Furthermore, these models enable a more accurate assessment of the potential impacts of varying dietary fatty acid (FA) profiles on obese-related dysmetabolic conditions.
Different dietary FA may play distinct roles in human obesity, particularly in the regulation of inflammatory signals and insulin resistance (IR). These differences may be related to variations in oxidation rates among FA, with long-chain saturated FA (SFA) being the least oxidized [
16]. Moreover, the bioactive metabolites derived from FA have the capacity to modulate lipid and energy metabolism by affecting peroxisome proliferator-activated receptor (PPAR)-α and endocannabinoid (EC) systems [
17,
18,
19]. These differences in FA impacts may partially explain variations in weight gain and the impaired lipid and energy metabolism observed in animals fed different types of dietary fat [
2,
20].
In vitro studies have shown that chronic treatment with palmitic acid (16:0, PA) impairs insulin secretion and promotes the formation of intracellular cholesterol, stearic acid (18:0), C16:0 dihydroceramide, and C24:1 sphingomyelin, leading to the decreased survival of pancreatic β-cells [
21]. Higher concentration of 16:0 may induce the expression of FA elongase 6 (Elovl6) [
22] and stearoyl-CoA desaturase (Δ-9-desaturase, SCD). The first enzyme converts 16:0 into 18:0, and SCD converts both to the respective monounsaturated (MUFA) palmitoleic acid (16:1n7) and oleic acid (18:1n9). High dietary intake of SFA has been associated with an increase in endogenous cholesterol synthesis and plasma lipoprotein cholesterol levels, potentially leading to lipotoxicity, cellular dysfunction, and the development of metabolic syndrome [
23]. Nonetheless, some randomized controlled trials failed to show an association between a reduction in SFA intake and lower total mortality or cardiovascular disease mortality [
24,
25]. The balance between dietary SFA/polyunsaturated fatty acids (PUFA), may well explain these contrasting results because of the importance of the correct amount of PUFA responsible for regulating serum cholesterol [
26,
27]. In fact, the role of dietary SFA seems to be closely dependent on their ratio with PUFA, and high levels of SFA, when accompanied by high levels of PUFA, do not appear to be associated with lipemic alterations [
28,
29]. Notably, a PUFA/SFA ratio of 3.75 and a MUFA/SFA ratio of 1.5 have been recommended for growth and maintenance formulations in rodent diets [
30], although a recent proposal suggests a revision of the FA composition [
30,
31].
Dietary FA intake can modify the composition of membrane phospholipid FA and their metabolites and lipid bioactive derivatives, such as EC and
N-acylethanolamines (NAE), which are involved in the homeostatic control of energy systems and lipid metabolism [
17,
18,
19]. Among the SFA, PA is the most implicated FA, with crucial physiological roles in several biological functions that are often overlooked. These seem to be closely related to the regulation of its tissue concentration, dependent on the balance of its endogenous biosynthesis and dietary intake [
32,
33]. This balance preserves the chemical–physical properties of membrane phospholipid, which can be influenced by the length and desaturation of the FA chain [
34]. A change in the tissue concentrations and metabolism of 16:0, along with its metabolites, 16:1 and 18:1, can affect various metabolic aspects of obesity and related disorders mediated by bioactive metabolites derived from membrane FA and, as such, influenced by dietary FA. However, it is often overlooked that increased tissue concentration of 16:0 and its metabolites in the form of 16:1 and 18:1 is primarily due to its biosynthesis through de novo lipogenesis, mainly from glucose in the liver and adipose tissue, rather than having a dietary origin [
32,
33].
Notably, most of the experimental animal studies aimed at evaluating the potential detrimental effects of 16:0 have been conducted using HFD, introducing an additional variable that does not translate directly to humans. Based on these premises, our study aimed to investigate the impact of a physiologically balanced dietary fat intake [
7] enriched in 16:0, but relatively low in linoleic acid (18:2n6) and α-linolenic acid (18:3n3), on IR, FA deposition, and metabolism in lean and obese Zucker rats. This dietary intake provides sufficient FA to prevent essential FA deficiency, with a PUFA/SFA ratio of 0.5, which is similar to the ratio observed in the Italian nutrient survey [
35] and typical of the Mediterranean diet. We compared this diet to the standard AIN-93G diet, which has a PUFA/SFA ratio of approximately 4:1.
4. Discussion
Our findings demonstrate that a reduced PUFA/SFA ratio did not affect food intake or weight gain, indicating that overnutrition-induced obesity in Zucker rats is independent of dietary FA composition within a physiological dietary fat content. An intriguing study by Jeffery and colleagues, in which weanling male Lewis rats were fed an HFD (178 g fat/kg) with varying proportions of 16:0, 18:1n9, 18:2n6, and 18:3n3, revealed that food intake did not vary among animals fed different diets. However, greater weight gain and higher final body weight were observed in rats fed diets with a low PUFA/SFA ratio (0.28 and 0.81) rich in 16:0 and containing low proportions of 18:1n9 [
48]. The contrast between these results and ours, which show no weight-gain differences due to dietary formulations, may be attributed to the high fat content used by Jeffery et al. compared to the normolipidic diet (despite the high 16:0 content) of our diet-PA.
In our study, there was a substantial increase in BMI in both groups of obese rats compared to lean rats, by 29% at the start of the study, and 57% in Ob-C and 36% in Ob-PA after eight weeks, with no discernible differences induced by diet composition. The BMI/Food Intake ratio, which can be considered an index of dietary energy substrate accumulation, predominantly fat, did not change after four weeks of dietary treatment. Yet, after eight weeks, lean and obese phenotypes were distinctly evident, irrespective of dietary treatment.
Body energy reserves are controlled by complex systems that regulate food intake, energy expenditure, and substrate partitioning. Therefore, an increase in the BMI/Food Intake ratio may signify excessive fat deposition leading to disrupted homeostasis in the obese phenotype, with the accumulation of ectopic fat in the liver, muscles, and plasma, and reduced metabolic flexibility, i.e., the capacity to efficiently utilize and store energy substrates based on fuel availability. This accumulation of energy substrates may be associated with an impairment related to their disposal, likely due to a metabolic inflexibility in obese rats, and may exacerbate glucose and lipid metabolism and promote inflammation. Metabolic inflexibility, which hinders the effective utilization of energy substrates for body growth, might account for the slightly shorter body and tail lengths observed in obese rats, regardless of diet.
Excessive dietary intake of SFA is usually associated with increased obesity-related hepatic inflammatory plasma markers such as ALT and AST [
49,
50,
51], as well as cytokines, such as TNFα and IL-1β, involved in the inflammatory response. Our results confirm that obesity induced liver damage and an inflammatory status; however, these markers were significantly reduced in Ob-PA rats compared to Ob-C rats.
Therefore, our data corroborate that obesity triggers systemic inflammation, lipid and glucose metabolic impairment, IR, de novo lipogenesis, steatosis, and liver damage. However, unexpectedly, we found that diet-PA improved insulin sensitivity, reduced inflammation, and mitigated liver damage in obese rats. These findings suggest that the harmful effects previously attributed to dietary PA may be due to the extremely high fat content rather than to PA itself.
We then explored possible mechanisms through which diet-PA exerted these positive effects in obese rats. The benefits do not seem to be directly associated with tissue PA concentrations. As already evidenced by others [
52], we did not observe an increase in tissue PA correlating with its dietary intake, likely due to a dilution effect, since 16:0 is, along with 18:1n9, the most abundant FA present in the body [
53]. In all tissues, we found an increase in PA related to obesity irrespective of the diet, except for a minor increase (18%) in the adipose tissue of Lean-PA. This suggests that the increase in 16:0 is more likely due to increased endogenous biosynthesis related to IR in obese rats [
54], rather than to its dietary intake. Chronic nutritional imbalance or pathophysiological conditions like obesity can strongly induce de novo lipogenesis in the liver and, to a lesser extent, in adipose tissue. This can lead to the overproduction of PA, resulting in an abnormal systemic inflammatory response and metabolic dysregulation, potentially causing dyslipidemia, IR, altered fat deposition, and other pathological conditions [
55].
Abdominal obesity predisposes to hepatic steatosis, via both the increased free fatty acid delivery to the liver and the hyperinsulinemia-induced increase in hepatic de novo lipogenesis [
56]. The sustained hepatic lipogenesis in obese rats was also evident from the increased 14:0 levels, similar to the 16:0 levels. In line with this, we observed an increase in liver weight and total hepatic lipids, indicating steatosis in obese rats, independent of dietary intake. This is likely related to obesity status, since lean rats fed diet-PA showed no further increase in hepatic lipid accumulation. While 16:0 and 16:1n7 were predominantly of endogenous origin, the 18:1n9 concentration was related to both dietary sources and endogenous biosynthesis. To maintain a stable 16:0 tissue concentration, its higher availability might induce prompt elongation and desaturation to 18:1n9 [
57,
58], preventing surplus accumulation. Consistently, 18:0 was significantly reduced in the livers of obese rats.
We assessed the impact of diet-PA on the tissue concentration of FA-related bioactive metabolites, such as EC and NAE, as these can modulate glucose and lipid metabolism through the PPAR-α and EC systems [
17,
18,
19], particularly under obesity conditions.
Diet-PA led to an increase in OEA levels in muscle, liver (trending), and adipose tissue in lean rats. OEA acts as a bioactive signal for the regulation of feeding and energy homeostasis, promoting satiety [
59], reducing lipogenesis [
60], and possessing analgesic and anti-inflammatory properties through the activation of PPAR-α [
61]. This nuclear receptor regulates appetite, food intake, energy homeostasis, lipid metabolism, and inhibits lipogenesis. Its activation may play a significant role in decreasing TG levels in plasma [
62], which may promote glucose homeostasis and insulin sensitivity. Dietary PA administration increased POEA levels in adipose tissue in both lean and obese rats, and in the liver and muscles of obese rats. Like OEA, this NAE is purported to act by binding PPAR-α, promoting increased FA oxidation and a reduction in inflammation [
63]. Interestingly, OEA and POEA also bind to the orphan G protein-coupled receptor 119 (GPR119), which has been shown to be able to stimulate the release of glucagon-like peptide-1 (GLP-1) from neuroendocrine cells [
64], thus improving insulin action. The precursor to POEA, 16:1n7, was notably increased in obese rats, most likely deriving from de novo lipogenesis, potentially masking that from PA desaturation of dietary origin. This FA is considered a lipokine that improves insulin sensitivity [
65,
66,
67].
Conversely, we found that different dietary concentrations of 18:2n6 substantially influenced its tissue levels and related desaturation and elongation metabolites. We thus investigated whether 18:2n6 tissue levels impact the concentrations of the EC AEA and 2-AG, both derivatives of 20:4n6, which may profoundly affect lipid and glucose metabolism. In human studies, 2-AG has been shown to positively correlate with decreased high-density lipoprotein cholesterol, and increased TG levels and IR [
68,
69]. It has been demonstrated that AEA and 2-AG levels were significantly elevated by a high dietary content of 18:2n6 in a low-fat diet and were associated with greater weight gain, adipogenicity, larger adipocytes, and macrophage infiltration in adipose tissue, compared to an isocaloric low 18:2n6 diet [
70]. In our study, 18:2n6 liver and muscle concentrations were positively correlated with 2-AG and AEA, and negatively correlated with PPAR-α ligands (OEA, PEA and POEA), suggesting that tissue 18:2n6 modulates bioactive metabolites favoring the predominance of the EC biosynthesis over PPAR-α ligands. Consequently, a reduced content of 18:2n6, as in diet-PA, led to a reduction in 2-AG levels compared to diet-C, confirming that a diet rich in 18:2n6 may favor an overactive EC system [
70].
We analyzed the OEA/2-AG ratio to provide an indication of the balance between the PPAR-α system and the EC system (supported by 2-AG). This balance may regulate the homeostatic control of energy metabolism and body composition. This ratio was elevated in rats fed diet-PA in muscle tissue, and also trended higher in liver and adipose tissue, suggesting the dominance of PPAR-α activity.
The PA-derived lipid mediator PEA exhibited a distribution pattern in the muscle similar to that of OEA, although it did not reach statistical significance. PEA is known to play an essential role in controlling the genesis of inflammation [
71] that may be exerted by acting as an agonist of the nuclear receptor PPAR-α and promoting the catabolism of proinflammatory eicosanoids by inducing peroxisomal β-oxidation [
59]. PPAR-α regulates the transcription of genes involved in the peroxisomal and microsomal oxidation of FA, thereby controlling serum levels of TG and cholesterol [
72,
73]. Furthermore, it has been shown that OEA and PEA improve metabolic flexibility [
43,
74]. This led us to investigate whether the balance between the PPAR-α and the EC system influences mitochondrial function, particularly in the liver and muscle.
In obese rats on diet-PA, we observed a beneficial impact on the restoration of mitochondrial respiratory activity, as evidenced by the heightened oxygen consumption rate with the FADH-linked (succinate) substrate. Notably, diet-PA had a pronounced effect on obese animals when we examined the mitochondrial FA oxidation rate using palmitoyl-carnitine as a substrate. Moreover, this increase in hepatic mitochondrial respiratory activity in Ob-PA rats did not result in an elevation in ROS production, unlike what was found in obese animals on diet-C. In fact, diet-C-fed obese rats displayed the highest H2O2 release and no variation in SOD activity.
Further, given the established role of skeletal muscle in metabolic flexibility, due to its association with mitochondrial dysfunction and IR [
75], we decided to evaluate the effects of diet-PA in modulating mitochondrial function in this tissue. Diet-PA led to an increase in mitochondrial FA oxidation rates both in lean and obese animals. This data led us to propose a metabolic shift towards the oxidation of FA in the skeletal muscle of diet-PA-fed animals.
Therefore, our data strongly suggest that the increase in muscle and liver of OEA, PEA and POEA, PPAR-α ligands, and the reduction in EC 2-AG, by enhancing the ratio between PPAR-α/EC system, might promote mitochondrial function and thereby improve glucose and lipid metabolism in dysmetabolic conditions such as obesity.
However, despite the significant results observed in circulating molecules related to IR and lipid impairment, and markers of inflammation and mitochondrial functions, we did not observe a reduction in weight gain or improvement in the altered depots of body energy substrate. We theorize that significant changes in body composition might require a longer dietary treatment, suggesting that changes in metabolic flexibility could precede possible changes in body fat deposition and distribution.
A significant improvement in glucose and lipid metabolism in obese conditions has been demonstrated to be greatly influenced by long-chain PUFAn3 20:5n3 and 22:6n3, particularly in the phospholipid form, and, more specifically, by the balance between n3/n6 [
38,
76]. Indeed, replacing lard, rich in SFA, with fish oil (rich in PUFAn3) in HFD can limit the development of systemic and tissue inflammation, reduce fat mass and IR associated with fat overnutrition, by modulating energy efficiency. In particular, at the skeletal muscle level, the PUFAn3-enriched diet promotes mitochondrial function and thereby metabolic flexibility [
29,
77].
Very low levels of 18:3n3 in diet-PA led to a systemic reduction in the n3HUFA score, which is considered a biomarker of n3 FA intake and tissue status, in all tissues in both lean and obese rats [
39]. Therefore, an addition of PUFAn3 to diet-PA might further improve glucose and lipid metabolism and metabolic flexibility. In practical terms, to reach an optimal 4:1 n6/n3 ratio in diet-PA, it would be sufficient to add 0.2% of 18:3n3 in the diet. A limitation of the present study, which could be addressed in future studies, is the absence of a group fed a diet with an intermediate PUFA/SFA ratio; indeed, we exclusively investigated diets with extremely low and high values of this ratio. Future studies could also explore the impact of diets containing varying proportions of MUFA and a higher n3/n6 PUFA ratio. It is worth noting that diets rich in PUFAn6 have been shown to inhibit the formation of highly PUFAn3 [
78], even though a recent comprehensive review suggests that augmenting n3, n6, or total PUFA has minimal or no effects on the prevention and treatment of type 2 diabetes mellitus [
79].