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Influence of dietary saturated fat content on adiposity, macrophage behavior, inflammation, and metabolism: composition matters.

We examined the effects of three high-fat diets (HFD), differing in the percentage of total calories from saturated fat (SF) (6%, 12%, and 24%) but identical in total fat (40%), on body composition, macrophage behavior, inflammation, and metabolic dysfunction in mice. Diets were administered for 16 weeks. Body composition and metabolism [glucose, insulin, triglycerides, LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), total cholesterol (TC)] were examined monthly. Adipose tissue (AT) expression of marker genes for M1 and M2 macrophages and inflammatory mediators [Toll-like receptor (TLR)-2, TLR-4, MCP-1, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, suppressor of cytokine signaling (SOCS)1, IFN-γ] was measured along with activation of nuclear factor kappa-B (NFκB), c-Jun N-terminal kinase (JNK), and p38- mitogen-activated protein kinase (MAPK). AT macrophage infiltration was examined using immunohistochemistry. Circulating MCP-1, IL-6, adiponectin, and leptin were also measured. SF content, independent of total fat, can profoundly affect adiposity, macrophage behavior, inflammation, and metabolic dysfunction. In general, the 12%-SF diet, most closely mimicking the standard American diet, led to the greatest adiposity, macrophage infiltration, and insulin resistance (IR), whereas the 6%-SF and 24%-SF diets produced lower levels of these variables, with the 24%-SF diet resulting in the least degree of IR and the highest TC/HDL-C ratio. Macrophage behavior, inflammation, and IR following HFD are heavily influenced by dietary SF content; however, these responses are not necessarily proportional to the SF percentage.


Metabolism
Plasma was assessed for fasting concentrations of glucose, insulin, total cholesterol (TC), HDL-cholesterol (HDL-C), and triglycerides at weeks 8, 12, 16, and 20, and for LDL-cholesterol (LDL-C) at weeks 16 and 20. Blood samples were collected from the tip of the tail after a 5 h fast. Blood glucose concentrations were determined in whole blood using a glucometer (Bayer Contour, Michawaka, IN). Collected blood was centrifuged and plasma was aliquoted and stored at Ϫ 80°C until analysis. Insulin concentrations were determined using an ELISA kit (Mercodia, Uppsala, Sweden), and colorimetric kits were used for plasma triglycerides (Pointe Scientifi c, Canton, MI), TC, HDL-C, and LDL-C (Genzyme, Kent, UK). Insulin resistance was estimated by the homeostatic model assessment (HOMA) index as follows: insulin resistance index = fasting insulin ( U/ml) × fasting glucose (mmol/l)/22.5 ( 20 ).

Tissue collection
At 20 weeks of age, mice were sacrifi ced for tissue collection. Epididymal, mesentery, and retroperitoneal fat pads were removed, weighed, and immediately snap-frozen in liquid nitrogen and stored at Ϫ 80°C or fi xed in 10% formalin until analysis. Blood was collected from the inferior vena cava using heparinized syringes, and then centrifuged at 4,000 rpm for 10 min at 4°C. Plasma was aliquoted and stored at Ϫ 80°C.

Adipocyte size and F4/80 immunohistochemistry
At sacrifi ce, a portion of epididymal AT was excised from each mouse, fi xed overnight in 10% formalin, dehydrated with alcohol, and embedded in wax. Paraffi n sections were stained with hematoxylin and eosin (H&E). The surface area of 100 adipocytes was determined (manual trace) and then averaged to represent mean adipocyte size for each mouse using Infi nity Analyze software (Lumenera, Ottawa, ON). F4/80 staining was performed in epididymal AT using rat monoclonal antibody (Serotec, Raleigh, NC). Color detection was visualized with a Vectastain avidin-biotinylated enzyme complex detection kit (R and D Systems, Minneapolis, MN), and 3,3 ′ -diaminobenzidine followed by counterstaining with hematoxylin.

Western blots
Epididymal AT was homogenized in radioimmunoprecipitation buffer (Sigma, St. Louis, MO), which included a protease inhibitor cocktail (Sigma, St. Louis, MO), and 1% glycerophosphate (100×), 0.5% sodium orthovanadate (1 mM), and 1% sodium fl uoride (5 mM). The protein concentration was determined by the Bradford method ( 21 ). Western blots were performed as previously described using primary antibodies for phosphorylated (Ser536) and total NFkB p65, phorphorylated (Thr183/Tyr185) and total JNK (Cell Signaling, Danvers, MA), and phosphorylated lean mice have an alternatively activated, anti-infl ammatory phenotype (M2) ( 7 ). These changes lead not only to increased infl ammation but also to dysregulation of metabolic homeostasis; infi ltration and polarization of macrophages in AT has been linked to lower plasma adiponectin levels as well as insulin and leptin resistance (8)(9)(10)(11). While the association between high-fat-diet-induced obesity and macrophage-mediated infl ammation has been clearly recognized, there is a fundamental gap in understanding the relative contribution of different types of fatty acids (FA) to these responses.
Saturated fatty acids (SFA) have received the most attention for their ability to infl uence pro-infl ammatory processes in high-fat-diet-induced obesity. These effects are thought to be largely mediated by their capacity to serve as ligands for Toll-like receptor (TLR)-2 and TLR-4; binding of SFAs to TLR-2 and/or TLR-4 on various cell types, in particular macrophages and adipocytes, results in the induction of proinfl ammatory gene transcription via activation of nuclear factor kappa-B (NF B), the c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) signaling cascades ( 10,12,13 ). Consistent activation of these pathways results in a chronic state of infl ammation and subsequent insulin resistance (IR) ( 10,14,15 ). In addition, SFAs, in general, are more obesogenic than other FAs. Long-chain saturated fatty acids (LCSFA, > C12:0) are not as effi ciently oxidized as unsaturated fatty acids (USFA) and thus are more likely to be stored as AT ( 16,17 ). Given the preponderance of evidence that supports a role of saturated fat (SF) on macrophage-mediated infl ammation and metabolic dysfunction in high-fat-diet-induced obesity, it is surprising that there have been no dose response studies to more clearly evaluate their specifi c role in these processes.
The purpose of this study was to examine the effects of three HFDs, differing in the percentage of total calories from SF (6%, 12%, and 24% of total caloric intake) but identical in total fat (40%), on body composition, macrophage behavior, infl ammation, and metabolic dysfunction in mice. We hypothesized that high dietary fat intake would increase adiposity, macrophage infi ltration, infl ammation, IR, and impair the lipid profi le, and that these effects would be augmented as the percentage of SF increased.

Animals
Male C57BL/6 mice were bred and cared for in the animal facility at the University of South Carolina. They were housed 4-5 animals per cage, were maintained on a 12:12 h light-dark cycle in a lowstress environment (22°C, 50% humidity, low noise), and were given food and water ad libitum. Principles of laboratory animal care were followed, and the Institutional Animal Care and Usage Committee of the University of South Carolina approved all experiments.

Statistical analysis
All data were analyzed using commercial software (SigmaStat, SPSS, Chicago, IL). Body weight, body composition outcomes, metabolic outcomes, the TC:HDL-C ratio, and the HOMA index were analyzed using a repeated measures two-way ANOVA. All other data were analyzed using a one-way ANOVA. Student-Newman-Keuls test was used for all posthoc analyses. Statistical signifi cance was set with an ␣ value of P р 0.05. Data are presented as mean ± SEM.

12%-SF consumption leads to heavier body weights, larger adipocyte size, and greater fat mass than any other diet
Body weights, fat pad weights, and average adipocyte size are presented in Fig. 1 . The mice consuming the 12%-SF diet had signifi cantly elevated body weights compared with control-diet-fed mice starting at 11 weeks of age ( P р 0.05), but this effect began later for 6%-SF and 24%-SF-fed mice (weeks 12 and 13 compared with AIN-76A and (Tyr182) and total p-38 MAPK (Santa Cruz Biotechnology Inc., Santa Cruz, CA) ( 22 ). As there were no differences in the activation of any of the measured proteins between the two control diets (AIN-76A and AIN-76A Mod), these samples were combined to represent the "control" diets for these analyses.

Concentration of circulating markers of obesity and infl ammation
Plasma concentrations of leptin, adiponectin, IL-6, and MCP-1 were determined using commercially available ELISA kits (R and D Systems, Minneapolis, MN). body fat percentages across the HFD-fed mice at 20 weeks ( P р 0.05). On the other hand, at week 20, the 12%-SF-fed mice had a greater fat mass than the 24%-SF and 6%-SF-fed mice ( P р 0.05). In general, lean mass increased over time for all groups, and by week 16, the HFD-fed mice exhibited greater lean mass compared with control-diet-fed mice ( P р 0.05).
It was not possible to calculate individual food intake, as four or fi ve mice were housed per cage. However, in general, we did not observe any differences among the HFDfed mice in weekly food intake (food consumed by mice in each cage/number of mice in cage) over the course of the study.
For all fat-pad depots, the HFD-fed mice had enhanced fat mass compared with control-diet-fed mice ( P р 0.05). Although there was no difference among HFDs with respect to total visceral AT, the 12%-SF-fed mice had a greater mesentery fat weight than both the 6%-SF and 24%-SF-fed mice ( P р 0.05).
Body composition analysis revealed a signifi cant difference among groups ( P р 0.05) ( Table 2 ). Specifi cally, the 12%-SF-fed mice had a greater body fat percentage compared with control-diet-fed mice (starting at 12 weeks) and 24%-SF-fed mice (16 weeks) ( P р 0.05). However, the 24%-SF and 6%-SF-fed mice did not differ from control-diet-fed mice until week 16. There were no signifi cant differences in

12%-SF diet leads to the greatest IR, followed by 6%-SF and 24%-SF diets, respectively
Beginning at week 16, all three HFDs produced higher fasting blood glucose concentrations compared with control diets ( P р 0.05), but by 20 weeks, only the 6%-SF and 12%-SF-fed mice had elevated fasting blood glucose concentrations ( P р 0.05) ( Table 3 ). And in fact, the 12%-SFfed mice exhibited a higher fasting blood glucose compared with the 24%-SF-fed mice at this time ( P р 0.05).
Similar to insulin, HFD-fed mice had a greater HOMA index than control-diet-fed mice at 16 weeks ( P р 0.05). However, at week 20, not only were the HFD groups different from the control-diet groups, but they were also different from each other; the 12%-SF groups had the greatest HOMA score, followed by 6%-SF and 24%-SF groups ( P р 0.05).

Changes in lipid profi le are infl uenced by SF content
The 12%-SF-fed mice had elevated TC compared with control-diet-fed mice at week 8 ( P р 0.05). By weeks 12 and 16, the 6%-SF and 12%-SF-fed mice had increased levels compared with the AIN-76A-fed mice (week 12) and AIN-76A-Mod-fed mice (week 16), respectively ( P р 0.05). All HFD-fed mice had a greater plasma concentration of TC versus the control-diet-fed mice at 20 weeks ( P р 0.05), but within the HFD groups, the 12%-SF group had significantly greater TC versus the 24%-SF group ( P р 0.05).
In general, the plasma HDL-C concentration tended to be highest with the consumption of the 24%-SF diet. Statistically, mice consuming the 24%-SF diet exhibited signifi cantly the consumption of the 12%-SF diet compared with all other diets, except the 6%-SF diet ( P р 0.05). We next confi rmed infl ammation and macrophage infi ltration in AT via H&E and immunohistochemistry staining of F4/80, respectively. The 12%-SF-fed mice showed increased infl ammation ( Fig. 2E ) and accumulation of macrophages in the AT ( Fig. 2F ) compared with control-diet-fed mice. Although the 6%-SF and 24%-SF-fed mice also exhibited increased infl ammation and macrophage infi ltration, these effects were not as pronounced as in 12%-SF.

Adipose tissue infl ammation is infl uenced by the content of dietary fat
There was no difference in the activation of p-38 MAPK among any of the diets ( Fig. 3A ). Alternatively, the activation of JNK, was signifi cantly increased in all HFDs ( P р 0.05) ( Fig. 3B ). However, only 12%-SF exhibited a higher degree of NF B p65 activation ( P р 0.05) ( Fig. 3C ).

Circulating leptin is greatest with the 12%-SF diet
For leptin, not only did all HFDs exhibit signifi cantly elevated plasma concentrations compared with control diets but there were also differences among HFDs; the 12%-SFfed mice had increased leptin levels compared with 6%-SF and 24%-SF-fed mice ( P < 0.05) ( Fig. 5D ). However, there were no signifi cant differences across the groups for circulating levels of IL-6, MCP-1, or adiponectin ( Fig. 5A-C ).  perturbations, ultimately leading to poorer health outcomes. It is well known that ATMs play a central role in this relationship ( 24 ). However, the extent to which the FA composition of a HFD infl uences macrophage behavior and infl ammation is still poorly understood; most of the available supporting literature is limited by the lack of control for various nutrients (e.g., ratio of MUFA:PUFA; omega-6:omega-3 FAs; protein:carbohydrate:fat, etc.), the utilization of a single ingredient as the sole source of dietary fat (e.g., corn oil, beef tallow, milk fat, etc.), and the absence of dose response studies. We examined the effect of three HFDs, differing in the percentage of total calories from SF (6%, 12%, and 24%) but identical in total fat (40%), on adiposity (absolute fat mass), macrophage phenotype, infl ammation, and metabolism utilizing controlled diets consisting of various lipid-rich ingredients. Our fi ndings indicate that manipulating the SF content without elevated HDL-C levels compared with the control-fed mice (week 8), 6%-SF-fed mice (week 16), and 12%-SF-mice (weeks 8, 16, and 20) ( P р 0.05). Interestingly, however, there were no signifi cant changes in HDL-C concentrations for any of the groups over time. Both the 6%-SF and 12%-SF-fed mice had a greater TC/HDL-C ratio than all other mice at week 20 ( P р 0.05). LDL-C, measured at weeks 16 and 20 only, was elevated in all HFD-fed mice compared with control-diet-fed mice ( P р 0.05). Differences in triglycerides across the groups were detected at 20 weeks only; the 24%-SF-fed mice and AIN-76A-Mod-fed mice had the highest and lowest levels of triglycerides, respectively ( P р 0.05)

DISCUSSION
HFDs are strongly linked with the accumulation of excess body fat, chronic inflammation, and metabolic Given that SFAs, in general, are less effi ciently oxidized than USFAs, we hypothesized that adiposity would be greatest following consumption of the 24%-SF diet ( 16,17 ). Interestingly, despite the fact that HFD-fed mice consumed similar kilocalories, the 12%-SF diet led to the greatest accumulation of fat and the largest adipocyte size, followed by the 6% and 24%-SF groups, respectively. We also confi rmed that plasma leptin was proportional to the degree of fat-mass accumulation as previously reported ( 9 ).
The FA composition of AT is primarily dependent on the FA composition of the diet ( 25 ). Therefore, lipolysis of white adipose tissue (WAT) composed of a greater proportion of SFAs should lead to higher levels of circulating saturated free-fatty acids and subsequent activation of macrophages through binding to TLRs ( 10,13 ). Given this, we expected that a diet higher in SF content would lead to greater macrophage infi ltration and more pronounced infl ammation. In agreement with previously reported literature, all HFDs increased expression of F4/80 as well as markers for M1 and M2 macrophages; interestingly, however, these reached statistical signifi cance only in the 12%-SF group. The activation of macrophages in AT is thought to be mediated by TLRs; both TLR-2 and TLR-4 have been implicated in HFD-induced macrophage activation and infl ammation given their ability to bind saturated free-fatty acids ( 13,26,27 ). Consistent with the macrophage data, we show a statistically signifi cant increase in TLR-2 expression only in the 12%-SF-fed mice, but surprisingly there was no signifi cant upregulation of TLR-4 in any of the HFD groups. To our knowledge, there have been no reports of an upregulation of WAT TLR-2 expression without signifi cant changes in WAT TLR-4 expression following HFD feedings. These results warrant further investigation into the role that individual TLRs play in HFD-induced infl ammation, and conversely, the effect that varying the composition of HFDs has on TLR activation.
Because macrophages are thought to mediate their infl ammatory processes through activation of various transcription factors ( 10,12,13 ), we next examined phosphorylated NF B, JNK, and p38 MAPK, and we found that all three HFDs increased activation of JNK, whereas only the 12%-SF diet signifi cantly increased NF B activation. All HFD-fedmice exhibited an increase in mRNA expression of MCP-1 in AT, and the 6%-SF and 12%-SF-fed mice, but not the 24%-SF-fed mice, exhibited an increase in TNF-␣ mRNA expression. Surprisingly, IFN-␥ , which has been shown to be upregulated in WAT and plays a key role in macrophage activation in HFD-induced obesity ( 28 ), was not statistically different across diets. This may be due to the specifi c measurement time point, the composition of these novel diets, or most likely, an increase in factors that can regulate expression of IFN-␥ . Additionally, we found no changes in AT IL-6 mRNA expression across groups. Although previous studies have shown AT IL-6 mRNA expression to be upregulated as a result of high-fat feeding ( 29,30 ), others have shown that this is not always the case ( 31 ). We also measured IL-10, an anti-infl ammatory cytokine, and found it to be upregulated in the 12%-SF mice only, which is changing the percentage of total calories from fat has a profound effect on these outcomes. The 12%-SF diet, most closely mimicking the standard American diet, led to the greatest adiposity (absolute fat mass), macrophage infi ltration, and IR. Although the 24%-SF diet increased adiposity and produced IR, it did not signifi cantly increase macrophage infi ltration, it led to a lesser degree of AT infl ammation, and it did not raise the TC/HDL-C ratio. explanation for the discrepancies between our fi ndings and those of others is the differences in the composition of the diets used.
Emerging evidence suggests that pro-infl ammatory M1 macrophages may play a role in inducing IR, whereas alternatively activated M2 macrophages may help to maintain insulin sensitivity ( 29 ). Our data somewhat supports this hypothesis as the degree of M1 macrophage mRNA expression corresponded well with the level of IR: the most likely a compensatory response to the increased infl ammation ( 32 ). Interestingly, SOCS1, a negative regulator of infl ammation ( 33 ), appeared to be downregulated in all the HFDs, but this reached signifi cance in the 24%-SF group only. Circulating markers of infl ammation (MCP-1, IL-6) were measured to determine whether they mirrored the observed changes in WAT as has previously been reported ( 30,34 ). We found no signifi cant increases in plasma MCP-1 or IL-6 for any of the HFDs. The most likely  plasma triglycerides, it also resulted in a higher HDL-C level, producing a more favorable TC/HDL-C ratio similar to that of control diets.
A possible explanation for the discrepancies in adiposity (absolute fat mass), IR, infl ammation, and the TC/HDL-C ratio between the 12%-SF-fed and the 24%-SF-fed mice may be the difference in the content of medium-chain fatty acids (MCFA, C8:0-C12:0) (3.6%, and 11.8% of total caloric intake for the 12%-SF and 24%-SF diets, respectively). These variations exist because it was not possible for the composition of the SF in each of the HFDs to be consistent while utilizing various lipid-rich ingredients and simultaneously controlling for the omega-6:omega-3 and MUFA:PUFA ratios. A previous study reported that a diet composed of 12% caprylic (C8:0) and capric acids (C10:0), 12%-SF diet resulted in the most severe IR, followed by the 6%-SF and 24%-SF diets, respectively. However, given that M1 macrophage quantifi cation in this study was limited to mRNA expression of CD11c, this association should be interpreted with caution. Adiponectin, well characterized as an anti-infl ammatory adipocytokine known to promote insulin sensitivity, has been shown to be inversely correlated with body fat accumulation ( 9 ). Interestingly, there were no changes in the concentration of plasma adiponectin across groups. It is likely that changes in adiponectin would have been observed if it had been measured in the WAT ( 35 ); however, our analysis was limited to plasma levels. Concerning lipid metabolism, all three HFDs increased TC and LDL-C levels. Of interest was the fi nding that although the 24%-SF diet increased LDL-C and elevated It is also likely that the differences in MCFAs, LCFAs, and linoleic content across the diets can explain some of the other unexpected reported fi ndings. For instance, the discrepancies in MCFA and linoleic content between the 6%-SF and 24%-SF diets may explain the similar adiposity but different levels of IR seen in these groups; previous work has shown that MCFA-rich diets can hinder the development of IR without infl uencing body weight ( 57 ). And it may be that the greater content of linoleic acid in the 6%-SF diet produced signifi cantly more 4-HNE, leading to a greater IR compared with the 24%-SF diet. Also, it is certainly possible that the relatively high content of both linoleic acid and LCSFAs in 12%-SF diet may explain the high degree of adiposity, macrophage infi ltration, and IR compared with the 6%-SF diet. Clearly, the disparate fi ndings across the three HFDs do not result from variations of a single group of FAs but instead stem from alterations in the content of several classes of FAs.
It is well established that SFAs play a role in infl ammatory signaling ( 10,12,58,59 ). However, the degree to which individual FAs activate pro-infl ammatory pathways remains somewhat controversial, as there has been at least one study to report that SFAs do not activate TLRs in vitro ( 60 ). Further, it is evident from our fi ndings as well as from previous research that varying the composition of dietary SFAs can differentially regulate infl ammatory processes in vivo compared with in vitro. For example, Lee and others have shown that lauric acid (C12:0), a MCFA that varies considerably with increasing percentage of SF in our diets, can serve as a potent agonist of TLR signaling in vitro ( 10,12 ). On the contrary, Rivera et. al. ( 46 ) reported that a MCFA-rich diet more effectively attenuated nonalcoholic steatohepatitis and reduced hepatic TLR-4 expression versus a PUFA-rich diet. These convergent fi ndings may be explained by the inclination of lauric acid to be oxidized in vivo, thus limiting its ability to serve as a ligand for TLRs ( 16 ). These fi ndings highlight the importance of additional research to better understand the role of various SFAs on infl ammatory-related signaling, and further, to determine whether the effects observed in vitro are actually refl ected in vivo.
Additionally, our data and that of others suggest that isocaloric diets with a greater content of omega-6 PUFAs can produce greater IR, result in a poorer TC/HDL-C ratio, and may even increase infl ammation ( 46,55 ), more so than an isocaloric diet composed of signifi cantly more SF. Meanwhile, others have shown that high consumption of omega-6 FAs has been associated with reduced infl ammation, a more favorable TC/HDL-C ratio ( 55 ), and no negative effects on infl ammatory markers ( 61,62 ). It should be noted, however, that the majority of these studies were performed in humans, in which it is extremely diffi cult to control for the nutrient composition of the diet and the activity level of the subjects--two factors known to greatly infl uence metabolism and infl ammation ( 63,64 ). This affi rms the need for future research utilizing various controlled diets to better understand the role that omega-6 FAs have on physiological processes. both MCFAs, augments the rate of fat-mass loss compared with a diet composed of 12% olive oil that contains mostly MUFAs ( 36 ). Furthermore, there is substantial evidence demonstrating that small doses of MCFAs and HFDs rich in MCFAs can effectively reduce body weight, reduce fatmass gains, and minimize IR compared with HFDs rich in LCFAs (37)(38)(39)(40)(41). Additionally, MCFA-rich diets have been associated with an improved cholesterol profi le (42)(43)(44)(45) and reduced infl ammation ( 46,47 ) compared with other isocaloric diets. The reported reduction in body weight produced by MCFAs is associated with higher energy expenditure and upregulated FA oxidation (48)(49)(50). As a result of their shorter chain length, MCFAs can be transported from the intestines directly to the liver where they can be quickly oxidized ( 37,51 ). LCFAs, on the other hand, are fi rst incorporated into chylomicrons before they leave the intestine via the lymphatic system and travel through the blood to extrahepatic tissues to be stored or metabolized. Not only do MCFAs and LCFAs differ in their digestive routes but their propensity to be oxidized is also dissimilar ( 16 ). Once inside a cell, MCFAs are less likely to be stored as AT as they can enter the mitochondria to be oxidized independent of carnitine palmitoyltransferase 1, unlike LCFAs.
It is important to point out that the MCFA argument may not be valid when comparing the 6%-SF and 12%-SF diets (0.1% versus 3.6% of total calories from MCFAs, respectively). It is likely that the MCFA-caloric content of the diet would need to be higher to produce similar effects generated by the 24%-SF diet. The differences in adiposity we observed between the 6%-SF and 12%-SF-fed mice may be due to the fact that the 12%-SF diet was composed of more obesogenic LCSFAs (8.4% versus 5.9% of total calories). However, we fi nd this unlikely as the 24%-SF diet had the largest percentage of LCSFAs (12.2% of total calories) and resulted in a similar level of adiposity as in the mice consuming the 6%-SF diet.
Another possible rationale for the differences in IR and infl ammation across the diets may be the disparities in the linoleic content (C18:2), (11.4%, 9.4%, and 5.4% of total caloric intake for the 6%-SF, 12%-SF, and 24%-SF diets, respectively). Even when controlling for various ratios within an isocaloric diet, the manipulation of one macronutrient, or subset of macronutrients, results in an uncontrollable alteration of another. As such, in the current study, alterations in the percentage of SF across diets also resulted in changes in the percentage of unsaturated fat. Thus, although the ratio of omega-6:omega-3 FAs was the same for each of the HFDs, the absolute quantity of linoleic acid in the 6%-SF and 12%-SF diets was greater than in the 24%-SF diet. Linoleic acid serves as a short-chain, parent omega-6 FA necessary for the synthesis of essential omega-6 LCFAs ( 52 ) that can play an important role in the promotion of infl ammatory processes through the production of various eicosanoids (52)(53)(54). In fact, others have shown that a HFD rich in omega-6 FAs can increase infl ammation more than a HFD rich in SFAs ( 55 ). Further, omega-6 FAs are prone to peroxidation, leading to accumulation of 4-hydroxy-2-nonenal (4-HNE), which has been shown to induce IR ( 56 ).
In summary, we examined the infl uence of three 40% HFDs, which differed in the percentage of total calories from SF (6%, 12%, and 24%), on body composition, macrophage behavior, infl ammation, and metabolic dysfunction in mice. In general, the 12%-SF diet, most closely mimicking the standard American diet, led to the greatest adiposity, macrophage infi ltration, and IR, whereas the 24%-SF diet had the lowest levels of these outcomes. In conclusion, our fi ndings support previously published data that ad libitum, high-fat feeding can lead to an increased risk of obesity and obesity-related side effects. However, the extent of excess fat accumulation and adverse health perturbations is not necessarily proportional to the percentage of SF in the diet. Although SFAs and omega-6 FAs have been implicated as pro-infl ammatory molecules, future research should examine the degree to which diets differing in SFA and omega-6 FA content impact individual TLR activation, macrophage behavior, infl ammatory signaling, and subsequent metabolic dysfunction, as well as the effect that manipulating the absolute quantities of omega-6 and omega-3 FAs, without changing the ratio between these FAs, has on these outcomes.