From saturated fat to arterial plaque: cholesterol and beyond

Cardiovascular diseases (CVDs) are still the leading cause of death globally (OWID), but vary markedly by demographics and in relation to various lifestyle factors. In particular, in the second half of the 20^th century, the seminal Seven Countries Study illuminated associations with diet, especially saturated fatty acids (SFAs), which at 50-year follow-up remain strongly associated with coronary heart disease mortality (n=16 cohorts, r=0.92) ¹. Many other studies have further probed this relationship, and while some heterogeneity emerged, so has context. For instance, some prospective cohort studies and meta-analyses thereof fail to find independent associations with SFA intake ²; however, studies performing substitution analyses generally report that replacing sources of SFAs with (plant and marine-sourced) unsaturated fatty acids (UFAs) or complex carbohydrates lowers CVD risk and mortality (e.g. US ^3–8, Europe ^9–11 and pooled ^12,13). Importantly, in the US it was also revealed that SFAs are typically replaced by refined grains and added sugars, which are also associated with CVD, potentially explaining prior null findings ⁴. In addition, dietary SFAs are also highly correlated with animal-sourced MUFAs (r=>0.8), which may have obscured favourable associations seen only with plant sources ^7,8.

Paralleling the observational research, a number of heterogeneous randomised controlled trials (RCTs) have also been conducted. In the latest Cochrane meta-analysis of such RCTs (n=15) lowering SFAs significantly lowered CVD events, mainly being driven by replacement with various PUFAs (n=8, RR=0.79, 95% CI 0.62–1.0) and carbohydrates (n=5, RR=0.84, 95% CI 0.67–1.06), but not CVD mortality (albeit with 75% less death vs. event data), and with no evidence of harmful effects (e.g. diabetes and cancer) ¹⁴. And in a more stringent analysis restricted to 4 core trials with PUFA replacement the results were even stronger (RR=0.71, 95% CI 0.62–0.81) ¹⁵. Consistent with this, a diverse literature of shorter RCTs show replacement of SFAs can favourably modulate various biomarkers of risk and pathophysiology, such as those relating to blood lipids ^16,17, metabolic health ^18,19, immunity ^20,21 and platelet/endothelial activity ^22,23.

Accordingly, lowering/replacing SFAs is a cornerstone of dietary guidelines worldwide, along with refined grains and added sugars (FAO). On the other hand, not everyone agrees ^24–27. Indeed as well as issues with food/nutrient replacements above, other nuances include the specific SFAs/PUFAs, food source/matrix, dietary patterns, endogenous biosynthesis (i.e. de novo lipogenesis, DNL) and genotypes, all of which may confer differential effects. And mechanistically, discussions are often limited to effects on plasma cholesterol, limiting biological plausibility. These factors in mind, this post explores some of the major biological pathways which may link dietary SFAs to CVD, as guided by qualitative comparisons with UFAs in humans and supported by preclinical models and mechanisms.

Atherogenesis

Most of the global CVD burden is due to ischemic vascular diseases involving atherosclerosis—i.e. plaque buildup in the arterial wall. Atherosclerosis starts in youth and progresses insidiously from initially superficial intima–media thickening, which increasingly protrudes the lumen and reduces blood flow, to eventual plaque rupture triggering thrombosis and infarction (e.g. heart attack or stroke). Importantly, atherosclerosis is a systemic disease and systemic imaging of general populations suggests subclinical plaque (i.e. stenosis or calcification) is present in around half of asymptomatic individuals by midlife (e.g. US ²⁸, Scotland ^29,30, Spain ^31,32 and Egypt ³³), affecting many arteries (esp. aorta–iliac) and correlating brain hypometabolism ³¹. At the cellular–molecular level, necropsy studies show the progression from initial fatty lesion to advanced plaque involves an increasing content of lipids, chiefly cholesterol (i.e. free and esterified) and phospholipids (i.e. phosphatidylcholines and sphingolipids) ³⁴, along with increasing leukocytes (esp. macrophages), necrosis, fibrosis and calcification ³⁵. The lipids are present in foam cells (i.e. as lipid droplets) and extracellular deposits (e.g. lipoprotein aggregates and crystals), while the simultaneous presence of apolipoprotein B (apoB) implicates plasma lipoproteins as a source ^34,36–38.

ApoB exists in 2 isoforms: apoB₁₀₀, or the truncated apoB₄₈, with a single copy of either being attached to lipoprotein particles exporting lipids from liver and intestine, respectively. In plasma the apoB₁₀₀-lipoprotein lineage dominates and is metabolised systemically, whereby lipolysis (via lipoprotein lipase, LpL) drives conversion of very-low-density lipoprotein (VLDL) to intermediate and low-density lipoprotein (LDL). Meanwhile apolipoprotein A-I (apoA-I) collects cholesterol from the periphery and matures into high-density lipoprotein (HDL) which transfers cholesteryl esters (CEs) to apoB-lipoproteins (via cholesteryl ester transfer protein, CETP), generating increasingly cholesterol-rich particles. Consequently, LDL is the major plasma cholesterol carrier, and with its relatively long plasma residence (in the order of days), the major apoB-lipoprotein. Ultimately, LDL is endocytosed via the LDL receptor (LDLR) and largely cleared by the liver ³⁹, where it subserves (indirect) reverse cholesterol transport; i.e. ~70% of HDL-CEs are transferred to VLDL/LDL before hepatic uptake ⁴⁰. However, the majority of peripheral cholesterol clearance may occur more rapidly via direct uptake of HDL-derived free cholesterol ^41,42. Once in the liver, cholesterol can be recycled and secreted back into plasma or catabolised to bile acids and excreted into faeces—fates which may be favoured by uptake of LDL and HDL, respectively ^43,44.

Conversely, in early atherogenesis lipids and lipoproteins accumulate in arterial intima at susceptible sites (e.g. branches and bifurcations) ^45–47, and prior to macrophages ³⁸. These sites are typically exposed to turbulent blood flow and shear stress and often exhibit increased lipoprotein permeability and/or retention ⁴⁸. Normally apoB-lipoproteins up to ~70nm in diameter (i.e. VLDL–LDLs and chylomicron remnants) can cross an intact endothelium, wherein binding to extracellular matrix proteoglycans (via electrostatic interaction) and LpL (acting as a bridge) promotes their retention ^38,45,46, while exposure to various enzymes and oxidants promote modifications, ultimately resulting in lipoprotein aggregation, fusion and cholesterol crystallisation ^37,49,50. A characteristic event of atherogenesis is the formation of foam cells, which contain abundant CE-rich lipid droplets, giving them their ‘foamy’ appearance. Lipoprotein aggregates isolated from human plaques induce accelerated macrophage uptake, greater cholesterol esterification ⁵¹ and inflammasome activation ³⁷. This reflects a dichotomy: foam cells can remove and digest harmful extracellular deposits liberating free cholesterol, with potential for efflux from arteries, but when overloaded can themselves be a site of crystallisation, inflammation and cytotoxicity ^47,52. In this regard, apoA-I/HDL isolated from human plaques is lipid-poor and pro-inflammatory, suggesting low acceptor/efflux activity ⁵³.

Cholesterol

Since the early 1900s, and the work of Anichkov, it was known feeding animals (e.g. rabbits) diets high in cholesterol can induce atherosclerosis ⁵⁴. Following this, early epidemiological studies (such as the Seven Countries Study) identified an association between serum cholesterol and CVD, which was later refined to LDL-cholesterol (LDL-c) ⁵⁵. To this day, studies explicitly on low risk populations show LDL-c can independently and linearly associate with subclinical atherosclerosis and CVD mortality ^32,56–58; even when including markers of LDL subspecies such as HbA1c (i.e. glycation), oxidised LDL (oxLDL) and lipoprotein(a) ³². However, in older cohorts associations with all-cause mortality may be inverted by malnutrition ⁵⁹. The causal role of LDL is supported by drug trials (i.e. late life lipid-lowering) and more recent Mendelian randomisation studies (i.e. lifetime lipids) ^55,60. However, CVD risk generally tracks better with apoB, which reflects the particle count of all atherogenic lipoproteins—with LDL typically representing >90%—and can be discordant from LDL-c (and non-HDL-c) due to variations in particle size/content, especially in metabolic disorders with abundant small-dense LDL particles (i.e. pattern B) ^61,62. Of note, such smaller particles exhibit various qualities which may render them particularly atherogenic ⁶³, albeit while carrying a smaller cholesterol load ⁶¹; indeed controlling for particle count, large and small LDL may similarly relate to CVD risk ⁶⁴. Moreover, recent Mendelian studies further suggest the risk from apoB is actually mediated by non-HDL-c ^65,66, which reflects the total cholesterol content of all apoB-lipoproteins. On the other hand, the inverse association between HDL-c and CVD seems non-causal, while cholesterol efflux capacity from macrophage to HDL predicts CVD risk better than HDL-c, suggesting HDL function is more important ⁴⁰.

Early cell studies suggested lipoproteins must first be modified in some way (e.g. oxidised) to promote atherogenesis—an enduring dogma. However, while lipoprotein modifications have many exacerbating effects discussed herein, this may not be strictly true. High levels of native LDL can dose-dependently induce macrophage foam cell formation in a non-saturable manner via receptor-independent fluid-phase pinocytosis ⁶⁷ and selective CE uptake ⁶⁸; as well as CCL20 secretion by vascular smooth muscle cells (causing lymphocyte migration) ⁶⁹. Atherogenic concentrations of LDL also induce endothelial dysfunction ⁷⁰ and permeability ⁷¹ (for reviews see ^52,72). In this regard, cholesterol crystals may form early in experimental atherosclerosis (i.e. before macrophage infiltration and neointima formation) via endothelial cells, which also process LDL but seem less able to upregulate efflux ⁷³. Such crystals can impair endothelial function (e.g. vasodilation, leukocyte barrier and cell survival) ^73,74, while activating endothelial and macrophage NLRP3 inflammasomes—a pathway required for atherogenesis, and which releases cytokines recruiting more immune cells ⁷⁵. Beyond the arterial wall, plasma lipids can also modulate extravascular pathways. For instance, hypercholesterolemia suppresses and lipid-lowering restores bone marrow-derived endothelial progenitor cells (EPCs), which mediate vascular repair ^72,76. Hypercholesterolemia also acts on bone marrow and peripheral cells to favour platelet biogenesis and activation ⁷⁷, as well as monocyte skewing and activation ⁷⁸, thereby priming the precursor to tissue macrophage foam cells ⁷⁹. Accordingly, cholesterol plays an important role in cell membrane signalling as discussed later.

In the Cochrane analysis of SFA-lowering trials above, meta-regression of various factors showed that the greater the reduction in serum cholesterol, the greater the reduction in CVD events, accounting for 99% of between-trial variation ¹⁴. Further, of these trials, STARS also measured angiographic progression of CAD, which correlated intake of animal-sourced trans fatty acids (TFAs) and SFAs (i.e. C14–18:0), and was also mediated via plasma cholesterol (except C18:0) ⁸⁰. Indeed it’s well established that isocaloric replacement of complex carbohydrates with common dietary SFAs (i.e. C12–16:0) raises total/LDL cholesterol, whereas typical plant-based PUFAs (i.e. C18:2/3) lower it, as tested in 100s of metabolic ward studies ¹⁶ and formularised since the 1950s ⁸¹. This dichotomy extends to low carbohydrate/ketogenic diets ^82–86; although more extreme hypercholesterolemia may relate to leanness and greater TG/VLDL turnover ⁸⁷ (even when favouring MUFAs ⁸⁸). The effect of SFAs may be accentuated by dietary cholesterol ^89,90 and attenuated in the case of cheese (vs. butter) ⁹¹. SFA-rich diets similarly increase apoB ^91–95 (for meta-regression see ¹⁷); more specifically, dairy fat/SFAs can increase all VLDL–LDL particles (vs. seed oils/n-6 PUFAs) ^92,96, large LDL (vs. MUFAs) ⁹⁴, or in people with pattern B, medium–small LDL particles (vs. MUFAs) ⁹⁵. These effects have been associated with increased CETP activity ⁹⁵ and decreased LDL catabolism ⁹⁷ and PBMC LDLR expression ^92,93 (e.g. %∆LDLR to %∆LDL-c r=–0.59 ⁹³). The response to SFAs (vs. carbohydrates) also depends on APOE genotype, being greatest with APOE4 (i.e. ~25% of Caucasians) ⁹⁸ and -219T promoter variants ⁹⁹; with the former also lowering the response to replacement with MUFAs ⁹⁸.

The atherogenic effect of SFAs is supported by non-human primate models ¹⁵. In particular, in African green monkeys fed cholesterol-containing diets with 35% fat for 5 years, palm oil/SFAs (vs. safflower oil/n-6 PUFAs) promote atherosclerosis, which correlates LDL-c and particle weight/size ¹⁰⁰. Rodent models are less representative of the human situation and typically employ genetic modifications to increase apoB-lipoproteins ⁵⁴, although still display differential effects and further inform underlying pathways. For instance, in mice with hepatic ablation of the LDLR a western diet enriched in butter/SFAs (vs. corn oil/n-6 PUFAs) induces hyperlipidemia and atherosclerosis ¹⁰¹. In normal and APOE^–/– mice injected with labelled human LDL a coconut oil/SFA-rich diet (vs. normal chow) increases arterial selective uptake of CEs, correlating plasma cholesterol, arterial LpL and atherosclerosis ¹⁰²; in later studies on LDLR^–/– mice incremental replacement with fish oil/n-3 PUFAs reduced hyperlipidemia, arterial macrophages/LpL and aortic lesions ^103,104. In LDLR^–/– mice circulating monocytes were also shown to internalise lipoproteins, become foamy-inflammatory and infiltrate nascent lesions ¹⁰⁵, which are reduced by replacing dairy fat/SFAs with plant-based UFAs (i.e. extra-virgin olive oil and nuts) ¹⁰⁶. However, in both monkeys and mice oleic/MUFA-rich diets can induce similar atherosclerosis to SFAs (vs. n-6/n-3 PUFAs), which especially correlates LDL particle size ^100,107. In mice this requires ACAT2 (aka. SOAT2) ¹⁰⁷ which synthesises oleate-rich CEs for apoB-lipoproteins ¹⁰⁸ and mediates LDL proteoglycan binding ¹⁰⁹ and aggregation ¹¹⁰. This contrasts the more favourable effects in humans of MUFA-rich diets on LDL size ^94,95 and binding ^109,111. Of note, animals may be fed higher cholesterol ^109,112, express higher ACAT2 (e.g. monkeys and rats) ^42,113 or have the LDLR knocked out (e.g. mice) ⁵⁴, each of which may increase MUFA sensitivity; while effects in humans may depend on food source ^7,8 and APOE variants ⁹⁸.

The ability of SFAs to reduce LDL clearance in animal models depends upon intake of dietary cholesterol ^112,114,115. In this regard, early rodent studies suggested UFAs (i.e. C18:1/18:2 UFAs vs. C12–16:0 SFAs) are better substrates for ACAT-dependent cholesterol esterification and so may lower free cholesterol in regulatory domains of the endoplasmic reticulum (ER) to induce SREBP-dependent LDLR expression ¹¹². However, in monkeys and rodents MUFAs stimulate the greater hepatic CE synthesis/secretion ^116,117 and LDLR expression ¹¹², suggesting other factors may underlie the greater lipid-lowering by n-6 PUFAs. Further, in a human tracer study (using radiolabelled mevalonic acid and free cholesterol) there was an absence of tissue CEs appearing in plasma, consistent with lower ACAT expression ⁴²; rather human plasma CEs are typically rich in n-6 PUFAs (i.e. C18:2 > C18:1 > C16:0) ⁸³, consistent with the phospholipid fatty acid preference of LCAT, which mediates esterification in plasma lipoproteins (esp. HDL). Accordingly, some human and rodent studies (but not all ¹¹⁸) find LCAT activity is modulated by dietary fat saturation (i.e. n-6 PUFAs > MUFAs > SFAs) and correlates plasma linoleate, while inversely with oleate ^119–121 and total cholesterol ¹²¹. Overexpression of human LCAT in transgenic rabbits also elevates HDL-c and lowers LDL-c via the LDLR, although this latter effect may involve high hepatic expression ¹²². Regardless, HDL-CEs are transferred to VLDL/LDL ⁴⁰ and UFAs may promote LDL–LDLR interaction. For instance, in men with hypercholesterolemia a 6-week walnut-rich diet increased LDL PUFA content and hepatocyte association in vitro, especially in those showing lower LDL-c and correlating C18:3 in core lipids (i.e. TGs + CEs) ¹²³. Following uptake of LDL, a greater availability of UFAs might also facilitate ACAT activity as above, and although this pathway may not influence the hepatic regulatory pool ⁴³, it may in non-hepatic cells ¹²⁴.

Changes to plasma cholesterol may also reflect hepatic excretion ^92,115. Early studies on systemic sterol balance generally found PUFAs (vs. SFAs) can increase faecal sterol excretion under non-steady state in normal adults, but not always those with familial hyperlipidemia, despite still lowering plasma cholesterol ¹²⁵. In the latter case this may be consistent with tissue redistribution ¹²⁶, as seen in guinea pigs (on low cholesterol diets for 6–7 weeks) ¹²⁷. Subsequent ileostomy studies showed isocaloric fat substitution with oils rich in PUFAs ¹²⁸ or MUFAs ¹²⁹ acutely increases net sterol excretion (within 2–4 days), which may be partly attributable to their phytosterol content ¹³⁰ (as controlled for in some earlier studies ^125,131). Several studies also implicate upstream changes to components of reverse cholesterol transport. For instance, healthy adults (n=122) with higher insulin resistance or SFA intake (>10% kcal) had lower ABCA1-dependent efflux to HDL (independent of HDL-c) ¹³². Also, replacing butter/SFAs with seed oils/n-6 PUFAs (without controlling for sterols) for 8 weeks induced serum bile acids as well as LXRα and ABCG1 transcripts in PMBCs, collectively suggesting increased efflux; and in multivariate analysis (incl. lipids, metabolites and gene expression) the most important explanatory variable was LXRα ⁹². Further, in hamsters decreasing the dietary fat saturation increased HDL–liver membrane binding (without affecting LCAT or CETP), which may explain the lowering of HDL-c with n-6 PUFAs ¹¹⁸. Regarding HDL function, in young healthy adults a single coconut oil/SFA-rich meal suppressed HDL anti-inflammatory activity (on endothelial cells) and flow-mediated dilation (FMD), while the former improved after safflower oil/n-6 PUFAs ¹³³. And in mice a palm oil/SFA-rich diet (vs. sunflower oil/MUFA, at 45% kcal) induced more body weight, liver lipids and inflammation, while lowering liver­–faeces cholesterol transport and enriching plasma HDL in acute-phase proteins ¹³². In summary, dietary UFAs (vs. SFAs) may lower plasma cholesterol largely by increasing tissue uptake (by liver and elsewhere), while also inducing reverse transport.

Metabolic

Recent decades have seen an epidemic of obesity and related diseases, including non-alcoholic fatty liver disease (NAFLD), metabolic syndrome (MetS) and type-2 diabetes (T2D), which are strongly associated with CVD and have shifted the typical risk factor profile toward increased serum triglycerides ¹³⁴, with attendant small-dense LDL. Triglyceride-rich lipoproteins (TRLs) also constitute non-LDL/HDL-associated ‘remnant cholesterol’ ¹³⁵, but may play a potent casual role in atherosclerosis beyond their cholesterol content ^66,136. Regarding diet, isocaloric replacement of carbohydrates with SFAs can lower serum triglycerides, but UFAs more so (i.e. C18-PUFAs > MUFAs > SFAs) ^15,17. While effects on postprandial lipemia seem equivocal (reviewed in ¹³⁷), SFA-rich meals (i.e. palm oil and cocoa butter vs. various UFAs) induced apoE expression on TRLs, which in vitro increased hepatocyte LDLR binding causing competitive inhibition of LDL uptake ¹³⁸; this effect was even greater in those with an APOE4 allele, which is itself associated with both CVD and Alzheimer’s disease ¹³⁹. Further, in mice a cocoa butter/SFA-rich diet (not MUFAs or fish oil/n-3 PUFAs) also induced amyloid-beta (Aβ) in gut enterocytes and plasma TRLs ^140,141, blood–brain barrier (BBB) dysfunction and Aβ transport to the brain ¹⁴²; which if corroborated in humans could underlie the epidemiologic association between SFA intake and Alzheimer’s ¹⁴³. Reciprocally, dementia and plasma Aβ₄₀ are associated with CVD ¹⁴⁴, while Aβ_40/42 binding to native or modified LDL enhanced foam cell formation in vitro ¹⁴⁵. In healthy adults high fat meals (vs. low fat) can also induce foamy-activated monocytes in association with postprandial TRLs ^146–148 and VLDL lipid saturation ¹⁴⁹, while TRLs from meals high in SFAs (vs. MUFAs and n-3 PUFAs) induce greater immune cell apoB₄₈ (chylomicron) receptor expression, lipid accumulation and activation ^150–152, and coronary smooth muscle cell invasion ¹⁵³. As chylomicrons are depleted of triglycerides they become remnant particles rich in cholesterol, which may particularly enter the hepatic regulatory pool to lower LDLR expression ⁴³. However, enrichment of chylomicron remnants with SFAs (vs. various UFAs) may lower hepatocyte LRP1 gene expression and uptake ¹³⁸, while inducing macrophage lipid accumulation ¹⁵⁴.

Serum triglycerides may also reflect systemic fat oxidation and storage. In human fatty acid tracer studies whole-body oxidation of typical dietary SFAs is lower than MUFAs and PUFAs, consistent with animal and cell studies ^155,156. Also, short-term low carbohydrate diets favouring UFAs (vs. SFAs) induce higher serum ketones ^82–84 (and improve long-term seizure control ⁸⁵), consistent with preclinical studies on hepatic β-oxidation and ketogenesis ^157,158. Furthermore, in short-term imaging trials on normal ¹⁵⁹ and overweight adults SFAs induce more liver fat (i.e. intrahepatic triglycerides) under isocaloric (i.e. butter/SFAs vs. sunflower oil/n-6 PUFAs ¹⁶⁰) or hypercaloric conditions (i.e. palm oil/SFAs vs. sunflower oil/n-6 PUFAs ^159,161; or various SFAs vs. UFAs and sugars ^162,163), while increasing the plasma SCD index (reflecting lipogenesis) ^159–161 and adipose lipolysis ¹⁶³, both of which may increase hepatic fatty acid availability. And inversely, in a trial on NAFLD patients randomised to specific diets (i.e. standard care, low carbohydrate or intermittent fasting) for 12 weeks, reductions in liver fat and stiffness correlated with increased plasma n-6 PUFAs and decreased intake of SFAs/MUFAs, respectively ¹⁶⁴.

Cardio-metabolic diseases typically involve insulin resistance and consequent hyperglycaemia, which is itself associated with CVD ¹⁶⁵; notably a 1-year RCT with insulin-stimulating drugs in T2D induced regression of carotid intima-media thickness (cIMT) in relation to postprandial glucose ¹⁶⁶. Even in the nondiabetic PESA cohort HbA1c (i.e. monthly glucose control) independently correlated the presence of subclinical atherosclerosis ³². Mechanistically, hyperglycaemia can induce oxidative-inflammatory activity and endothelial dysfunction ¹⁶⁵, while glycation of LDL increases arterial proteoglycan binding ^167,168. Insulin itself also promotes hepatic apoB metabolism ¹⁶⁹ and may affect many atherogenic cells ^170,171. Regarding diet, in a systematic review and meta-analysis of controlled feeding trials isocaloric replacement of SFAs with PUFAs improved insulin sensitivity and glucose control ¹⁸. Further, in people spanning the range of insulin sensitivity (i.e. athletes–lean–obese–T2D), intramuscular accumulation and subcellular localisation (i.e. sarcolemma and organelles) of saturated triglycerides ¹⁷² and sphingolipids/ceramides ¹⁷³ correlate insulin resistance, while SFA intake was increased in T2D ¹⁷². Accordingly, in some trials on healthy adults a higher palmitate/SFA intake (vs. oleate/MUFA) for 2–3 weeks ^174,175, or as a single bolus ¹⁷⁶, induced blood/muscle sphingolipids/ceramides and suppressed glucose metabolism and insulin sensitivity. Also, in trials on overweight adults over-feeding SFAs from mixed sources (vs. UFAs or sugars) for 3 weeks ¹⁶³, or palm oil/SFAs (vs. sunflower oil/n-6 PUFAs) for 8 weeks ¹⁶¹, increases multiple plasma/LDL sphingolipid species (opposite to PUFAs), paralleling induction of insulin resistance and liver fat. And inversely, in obese adolescents on a 1-year multidisciplinary intervention, SFA reduction correlated a decreased insulin and increased adiponectin/leptin ratio, which itself negatively correlated cIMT ¹⁷⁷. Of note, in mice and human cells palm oil/SFAs (vs. olive oil/MUFAs) also induced intestinal insulin resistance via ceramide, thereby impairing the ability of insulin to inhibit triglyceride secretion and linking to postprandial hypertriglyceridemia ¹⁷⁸.

Besides glucose-insulin homeostasis, sphingolipids may have more direct effects on atherogenesis. Indeed alongside cholesterol, atherosclerotic plaque was long known to contain sphingolipids ³⁴. More recently various sphingomyelins and ceramides were identified and associated with plaque inflammation and apoptosis ¹⁷⁹, while serum ceramides (esp. Cer16:0, Cer18:0 and Cer24:1) predict CVD risk independent of conventional risk factors (incl. apoB) ¹⁸⁰. Serum ceramides are particularly elevated in obesity and T2D ¹⁸⁰; although LDL ceramides were only elevated in the latter and in preclinical models induce macrophage activation and muscle insulin resistance ¹⁸¹, involving mitochondrial dysfunction ¹⁸². Further, LDL can deliver ceramide to endothelial cells ¹⁸³, where it can mediate apoptosis ¹⁸³, suppress nitric oxide (i.e. eNOS) ¹⁸⁴ and increase the uptake and retention of oxLDL ¹⁸⁵. Moreover, aggregated LDL from human plaques was highly enriched in ceramide, a product of sphingomyelin cleavage by SMase, which promotes LDL aggregation and fusion in vitro ⁴⁹. Accordingly, LDL susceptibility to aggregation, as induced by human secretory sphingomyelinase (S-SMase), was associated with CVD death independent of traditional markers (e.g. LDL-c) and activated macrophages and T cells in vitro (contrasting oxLDL) ¹¹⁰. LDL aggregation was also related to the surface/core lipidome (esp. ↑ sphingolipids/ceramides vs. phospholipids) and favourably modified by a ‘Healthy Nordic diet’ (incl. ↓ SFA/PUFA ratio; Fig. S5) or lipid-lowering drug (i.e. PCSK9 inhibition) ¹¹⁰. In a subsequent trial on overweight adults over-eating SFAs from mixed sources for 3 weeks induced LDL sphingolipids and aggregation, while UFAs (i.e. 57% MUFAs, 22% PUFAs) lowered LDL proteoglycan binding (and apoE) and sugars were without effect ¹¹¹. A further study including liver biopsies reported that LDL aggregation and lipid composition correlates the liver lipidome, implicating hepatic sphingolipid metabolism in LDL composition ¹⁸⁶. In rodent models high fat diets induce de novo sphingolipid synthesis (i.e. via SPT) and salvage pathway turnover increasing the generation of long-chain ceramides in liver, plasma and elsewhere, while the SPT inhibitor myriocin ameliorates atherosclerosis (reviewed in ¹⁸⁷). Also, in LDLR^–/– mice a diet rich in cholesterol and dairy fat/SFAs induced macrophage S-SMase, which acts on serum LDL to increase ceramide and susceptibility to aggregation and oxidation ¹⁸⁸.

Oxidation

OxLDL is present in plaques and plasma where it’s associated with CVD ^189,190, although not always independently of apoB (e.g. CHD ¹⁹¹ and MetS ¹⁹²), likely due to 4E6 antibody cross-reactivity ¹⁹³. On the other hand, oxidised phospholipids on apoB₁₀₀ (oxPL–apoB) are independently associated with CVD and mainly carried by Lp(a), an LDL variant; indeed oxLDL donates its oxPL to Lp(a) in vitro ¹⁹³. OxLDL normally represents a very small fraction of plasma LDL ¹⁹⁴ and increases preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ¹⁹⁵; similar to oxPL–apoB ¹⁹³. Oxidation of LDL in vitro is typically achieved by incubation with copper (Cu²⁺) sulfate solution or arterial cells cultured in media containing transition metals, which induce 1-electron oxidations via Fenton chemistry. This enhances its pro-atherogenic endothelial/inflammatory effects (e.g. eNOS ⁵², CCL20 ⁶⁹, EPCs ⁷² and HSPCs ¹⁹⁶) and induces macrophage uptake via scavenger receptors ¹⁹⁰. Here it induces lipid droplets, which may be limited by defective lysosomal processing ⁶⁷, and also lysosomal crystals and NLRP3 activation ^75,197. As the major transition metal in vivo, iron (Fe²⁺) dysregulation may particularly promote oxidation during plaque haemolysis ¹⁹⁸. Moreover, many studies also report that human plaques have increased expression of inducible nitric oxide synthase (iNOS) ^199–203 and myeloperoxidase (MPO) ²⁰⁴ (which employ a central heme-iron active site to generate oxidants), while recovered LDL and apoA-I/HDL are highly enriched in their 2-electron protein oxidation products (i.e. nitrotyrosine and 3-chlorotyrosine, respectively) ^205,206, implicating immune-dependant redox modifications ²⁰⁷. These oxidations are not blocked by serum (unlike copper oxidation) and resulting NO₂–LDL stimulates macrophage uptake and cholesterol loading via scavenger receptor CD36 ^208,209. Note however, in early studies LDL isolated from human aortic fatty streaks and plaques was not sufficiently oxidised for receptor-mediated uptake, which instead was increased in a non-saturable manner attributable to aggregates ⁵¹. Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^51,68 and CE accumulation beyond native or oxLDL ^210,211. Further, mildly oxidised LDL inhibits native LDL-induced foam cell formation ²¹², although such particles tend to aggregate ⁴⁹ and still induce macrophage crystals ^75,197, so may contribute in this way ⁵¹.

Of lipids PUFAs are particularly susceptible to oxidation, of which linoleic acid (C18:2n-6) is most abundant in LDL and plaque CEs. Accordingly, electrospray MS/MS revealed a substantial proportion of peripheral plaque CEs are oxidised, and cholesteryl linoleate to the greatest extent ²¹³. This may occur despite normal levels of the major lipophilic antioxidant α-tocopherol (aka. vitamin E) ^214,215; i.e. contrasting typical conditions in vitro and consistent with failed antioxidant trials ²⁰⁷. Importantly, while under strong copper-oxidising conditions α-tocopherol acts as a chain-breaking antioxidant and underlies the lipid oxidation lag phase, under more mild conditions the α-tocopherol radical can initiate lipid oxidation ²¹⁶, or when there are insufficient regenerative co-antioxidants (e.g. CoQ₁₀ and carotenoids) ²¹⁷; and α-tocopherol does not block iNOS/MPO-derived oxidants ²¹⁴. Further, human aortic lesions from early to end-stage disease had accumulation of cholesterol (stages II–III) and CEs (stages IV–V) before their major oxidised derivatives (i.e. 27-hydroxycholesterol and CE hydroperoxides/hydroxides, respectively), while α-tocopherol and CoQ₁₀ levels remained stable, consistent with mild and enzymatic oxidative responses to lipid accumulation ²¹⁸. Indeed 27-hydroxycholesterol is produced by sterol 27-hydroxylase and this pathway may facilitate cholesterol efflux, especially when HDL is deficient ²¹⁹. As above, apoA-I/HDL isolated from human plaques is lipid-poor and oxidised by MPO, wherein modified tryptophan residues inactivate its ABCA1-dependent acceptor activity ^206,220. More recent high-resolution imaging of advanced carotid plaques has also revealed that oxidised CEs co-localise with sphingomyelin in the necrotic core ²²¹. Of potential relevance, 1-electron oxidation of LDL-c generates 7-ketocholesterol, which in macrophages inhibits lysosomal sphingomyelinase (SMase) causing accumulation of sphingomyelin–cholesterol particles ²²², and also dose-dependently induces cholesterol crystals ²²³.

Regarding diet, in various short-term trials plant-based MUFA-rich diets can lower LDL and HDL oxidation, and susceptibility to copper oxidation (i.e. lag time and/or rate) and monocyte adhesion in vitro, correlating oleate/linoleate ratios and opposite to n-6/n-3 PUFA-rich diets ^224–226. However, this doesn’t parallel favourable associations between PUFAs and hard outcomes or factor other important precursors to oxidation in vivo, such as arterial lipoprotein retention and inflammation, as above. Accordingly, in men ²²⁷ and monkeys ¹⁰⁰ n-6 PUFA-rich diets increase linoleate/oleate ratios in plasma and plaques (and LDL susceptibility to oxidation ¹⁰⁰), yet are protective; the long-chain n-3 content of advanced carotid plaques is also increased by supplementation and correlates greater stability and lower inflammation, consistent with anti-inflammatory effects ^228,229. Furthermore, the food matrix is also important. For instance, in healthy adults an n-6/n-3 PUFA-rich walnut meal (i.e. 59g fat, 42g PUFAs) increased postprandial antioxidant capacity and lowered MDA (5-hour AUC) and oxLDL (at 2 hours) ²³⁰, while longer trials show enrichment of PUFAs with preservation of oxidation status ^231,232, alongside many other cardio-protective effects (reviewed in ²³³). Conversely, red meat and heme-iron intake are associated with CVD ^234–237 and can promote lipid oxidation during digestion ²³⁸. For instance, in humans and mice red meat ingestion induced postprandial plasma lipid oxidation and corresponding LDL-MDA modification, which was greatly inhibited by polyphenols ^239,240; and in gastric models olive oil/MUFAs inhibited red meat/iron-induced lipid peroxidation, opposite to fish oil/n-3 PUFAs ²⁴¹. Of note, in a unique RCT comparing oxidised vs. high quality fish oil, only the latter lowered apoB-lipoproteins ²⁴²; indeed excessive PUFA oxidation may eventually abrogate their benefits. Some studies also suggest excess dairy fat can induce oxidative stress in vivo. For instance, in healthy adults a high fat milkshake (vs. low fat) induced pathological RBC remodelling and foamy monocytes, while elevating plasma and RBC-bound MPO in association with impaired FMD and chlorination of HDL; tested in vitro major cow milk fatty acids (i.e. oleic or palmitic acid) induced MPO release by monocytes and uptake by porcine arteries ¹⁴⁶. In mice a diet rich in dairy fat/SFAs also elevated oxidised HDL and LDL, while replacement with soybean oil/PUFAs (i.e. ~5:1 of n-6:n-3) enhanced HDL antioxidant activity ²⁴³; and in another study replacement with olive oil and nuts lowered monocyte oxLDL uptake and CD36 expression, which was modulated correspondingly by TRLs on each diet ¹⁰⁶.

Lipopolysaccharides

In 1999 the prospective Bruneck study of older Italians (n=516; age 50–79 at baseline) published the first evidence of an association between circulating levels of lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, and early carotid atherosclerosis, which was independent of traditional vascular risk factors (incl. apoB) except smoking ²⁴⁴. Nowadays many studies support a link with cardiometabolic disease and athero-thrombosis (e.g. reviews ^245–247). LPS is the canonical ligand for toll-like receptor 4 (TLR4), which stimulates innate immunity and primes the NLRP3 pathway ^37,75,197; although depending on source/structure, it can act as an agonist or antagonist (e.g. E. coli and Bacteroides, respectively) ²⁴⁸. In mouse models of endotoxemia, platelet TLR4 triggers neutrophil extracellular traps (NETs) to ensnare bacteria in liver sinusoids and pulmonary capillaries ^249,250, but which also promote monocyte recruitment and aortic atherosclerosis ^251,252, and increase carotid plaque MPO and instability ²⁵³, consistent with human samples ^252,253. In particular, E. coli-LPS was present in human carotid plaques (esp. necrotic core; Fig. 1A) and associated with enlarged macrophages; tested in vitro similar LPS levels incubated with monocytes induced TLR4-dependent NADPH oxidase 2 (Nox2) and oxLDL ²⁵⁴. Further, carotid LPS correlated plasma LPS (r=0.668), which correlated soluble TLR4 and serum zonulin (a marker of intestinal permeability) ²⁵⁴; with similar blood marker relationships reported in other populations ^255–257. Accordingly, several gut microbiome studies on people with CVD find elevated Gram-negative Enterobacteriaceae (e.g. E. coli and Klebsiella) and decreased butyrate-producing bacteria (e.g. Roseburia and Faecalibacterium) ^258–260. Butyrate is the archetypal beneficial short-chain fatty acid (SCFA) and ameliorates atherosclerosis in APOE^–/– mice by lowering gut permeability and endotoxemia ²⁶¹ and inducing ABCA1-dependent cholesterol efflux ²⁶². Several human studies also find depletion of Bacteroides spp. ^258,263,264, which when administered to APOE^–/– mice also lowered atherosclerosis and gut/blood LPS ²⁶³.

The systemic transport of exogenous (microbial) lipids is analogous to that of endogenous lipids. Indeed in blood both LPS and Gram-positive lipoteichoic acid (LTA) are largely bound to lipoproteins ²⁶⁵, transferred from HDL to LDL (via LBP and PLTP) ^266,267, and removed predominantly via the hepatic LDLR (in humans) ^268,269. A single LDL particle can bind many LPS molecules with only minor changes to its composition ²⁷⁰; such binding sequesters the lipid A region within the phospholipid monolayer and hepatic uptake is apparently non-toxic ^268,269. Accordingly, human PCSK9 loss-of-function variants, which increase LDLR expression and lower plasma LDL, were associated with improved sepsis survival and lower LPS-induced inflammation in vivo ²⁷¹, contrasting the situation in LDLR^–/– mice ^271,272. Moreover, lipoproteins may also carry bioactive LPS into other tissues to elicit inflammation (e.g. endothelium ^273,274, adipose ^275,276 and brain ^277,278), which would presumably be facilitated by slower hepatic uptake. In particular, LDL–LPS complexes formed in vitro acquire a negative charge and have increased binding and accumulation in arterial wall and macrophages ²⁷⁹. Here LPS can induce smooth muscle cell synthesis of elongated proteoglycans (equal to traditional agonists) ²⁸⁰, increase LDL susceptibility to oxidation (by copper, endothelial and smooth muscle cells) ²⁸¹, and stimulate macrophage oxLDL uptake and foam cell formation ²⁸²; while at concentrations found in CAD, LPS and indoxyl sulfate (a microbial metabolite of tryptophan) exhibited co-toxicity on endothelial cells ²⁶⁴. Moreover, in humans and rodents LPS impairs total (i.e. macrophage–faeces) reverse cholesterol transport at multiple steps ²⁸³; in part via induction of MPO/SAA ²⁸⁴ and suppression of ABCA1 ²⁸⁵.

In another prospective study on people with atrial fibrillation (n=912; mean age 73.5), blood LPS was associated with major CVD events, platelet activation and LDL-c, and negatively with Mediterranean diet scores (esp. fruit and legumes) ²⁸⁶. Dietary fats also modulate gut bacteria and plasma LPS. For instance, in a 3-week trial on overweight adults (similar to those above ^111,163), over-feeding SFAs increased faecal Gram-negative Proteobacteria ¹⁶³, while UFAs increased butyrate-producing bacteria (i.e. Lachnospira, Roseburia and Ruminococcaceae spp.) ¹⁶². Similarly, in a double-blind crossover trial, replacement of butter/SFAs with margarine/n-6 PUFAs for just 3 days induced Lachnospiraceae and Bifidobacteria, with the former negatively correlating total cholesterol (r=­–0.511) ²⁸⁷. While not generally altered in CVD ²⁵⁸, SFA-rich diets may also elevate bile-resistant, sulfide-producing genera (i.e. esp. Bilophila; in faeces ^162,288 and mucosa ²⁸⁹), which in mice results from increased secretion of taurine-conjugated bile acids ²⁹⁰ and lowers colonic butyrate and barrier function ^290–292. Moreover, in a systematic review of RCTs assessing the effect of fat quality on metabolic endotoxemia, SFA-rich meals can increase postprandial LPS in both normal and overweight subjects ²¹. For instance, in healthy adults a 35% fat porridge meal made with coconut oil/SFAs increased postprandial LPS (vs. fish oil/n-3 PUFAs), but not serum cytokines, whereas grapeseed oil/n-6 PUFAs did not ²⁹³; although an intermediate effect was significant in a prior pig study ²⁹⁴. Further, in other trials dairy fat (vs. carbs, MUFAs and n-3 PUFAs) increased postprandial LPS alongside PBMC activation (incl. TLR2/4) and endothelial adhesion markers ^295–297; an effect quicker in obesity ²⁹⁷ and still present after 12 weeks of a SFA-rich diet ²⁹⁶. Also, in people with impaired fasting glucose postprandial LPS correlated apoB₄₈ (i.e. chylomicrons) and oxLDL, which was suppressed by substituting cheese/SFAs for extra-virgin olive oil/MUFAs, and inversely correlated plasma polyphenols; tested in vitro equivalent LPS levels incubated with platelets induced TLR4-dependent Nox2 and oxLDL ²⁹⁸. Accordingly, in preclinical studies LPS absorption can occur via chylomicrons ²⁹⁹, which deliver bioactive LPS to lymph ³⁰⁰. Short-term controlled-feeding crossover trials on healthy adults also report that lowering a habitual western diet palmitate/oleate ratio (from ~1:1 to 1:10) lowers LPS-induced cytokine secretion in vitro ^301–303. So taken together, SFA-rich diets may increase LPS biosynthesis, translocation and sensitivity, while potentially lowering LDLR-mediated hepatic clearance.

In human and experimental fatty liver, hepatocyte LPS is also increased, associated with immune-inflammatory markers ³⁰⁴ and implicated in pathogenesis (e.g. reviewed in ³⁰⁵). As above, SFA-rich diets can induce liver fat; in particular, one group found this occurred alongside increased adipose lipolysis and inflammation (i.e. tissue transcriptome), blood ceramides, liver enzymes and endotoxemia (i.e. LBP/CD14 ratio), faecal Proteobacteria ¹⁶³ and baseline abundance of Bilophila ¹⁶². Accordingly, in mice Bilophila wadsworthia aggravates dairy fat/SFA-induced metabolic dysfunctions and steatosis, while suppressing microbial butyrate and promoting LPS biosynthesis and translocation ²⁹¹. In the postprandial setting, a trial on healthy adults given a single bolus of palm oil/SFAs (equivalent to a SFA-rich meal) induced whole-body/adipose/liver insulin resistance, while elevating intrahepatic triglycerides and plasma free fatty acids, but not inflammatory markers; although a parallel mouse study revealed transcriptomic evidence of hepatic LPS/TLR signalling ³⁰⁶. Of importance here, fat-induced LPS absorption may not only involve chylomicrons, but a more rapid and dominant portal vein pathway ³⁰⁷, wherein intestinal secretion of HDL₃ binds LPS and restrains high fat/lard-induced liver injury and fat storage ³⁰⁸. For further context, in other postprandial trials cream-induced liver fat was attenuated by co-administration of 50g glucose but not fructose ³⁰⁹, which itself may also be capable of inducing endotoxemia ³¹⁰, DNL and ceramides ³¹¹. This unique effect of glucose may involve its ability to stimulate insulin and thereby inhibit adipose lipolysis and fatty acid flux to the liver ³⁰⁹. Similarly, in humans experimental endotoxemia induces peripheral inflammation, oxidative stress and lipolysis, the latter 2 of which were particularly inhibited by co-infusion of insulin ³¹². Such endotoxemia also induces ceramides in VLDL and LDL ³¹³, which in rodents is accompanied by activation of S-SMase in serum and de novo sphingolipid biosynthesis (i.e. SPT) in liver ^313,314. Therefore LPS may mediate some of the differential effects of SFAs above.

Microdomains

The body is an aqueous environment and lipids are stored and transported in amphipathic membranes which exhibit heterogeneous biophysical properties in relation to their specific compositions. The plasma membrane exists largely in a state of liquid-disorder (L_d) with distinct liquid-order (L_o) microdomains (aka. lipid rafts) which are characteristically detergent-resistant and enriched in cholesterol and saturated sphingolipids ³¹⁵. Such rafts may serve as functional platforms to assemble proteins subserving cell signalling and endocytosis ³¹⁶, which can be modulated by exogenous lipids. In particular, free cholesterol in the lipoprotein monolayer is in equilibrium exchange with cell membranes ³¹⁷, while VLDL and LDL were reported to preferentially interact with model membrane raft regions, consistent with the high affinity of apoB₁₀₀ for cholesterol (and contrasting triglyceride-rich chylomicrons) ³¹⁸. Further, the hepatic LDLR is associated with both clathrin and caveolae-rich membrane regions ³¹⁹ (which correspond to non-raft and raft regions, respectively ³¹⁶), and treatment with LDL or cholesterol induced translocation to caveolae coinciding with reduced LDL uptake ³¹⁹. In non-hepatic cells ⁴³ internalised LDL-c also travels from lysosomes to plasma membrane first before ER regulatory domains ¹²⁴. And in the other direction, the ABCA1 transporter may associate with cholesterol-rich lipid rafts ³²⁰ to mediate efflux to apoA-I/HDL ³²¹; indeed the composition of nascent HDL resembles lipid rafts ³²¹. Therefore conditions of high membrane cholesterol may favour reduced uptake and increased efflux. Membrane fluidity is also determined by lipid saturation, with L_d regions containing phospholipids enriched in UFAs. Challenging cells with PUFAs results in rapid plasma membrane incorporation and compensatory induction of saturated lipids and cholesterol to maintain biophysical homeostasis ³²². Conversely, exogenous SFAs induced accumulation of saturated glycerolipids in the ER and solid phase (i.e. solid-order, S_o) membrane separation in a manner correlating SFA chain length and offset by UFAs, which may instead promote channelling into lipid droplets ³²³. In monkeys corn oil/n-6 PUFAs (vs. coconut oil/SFAs) increased LDL uptake by PBMCs which correlated membrane fluidity and lower plasma cholesterol ³²⁴; while in vitro enrichment of hepatocytes in various fatty acids affected LDL binding/metabolism and membrane fluidity in a highly correlated manner (i.e. n-6 PUFAs > MUFAs > SFAs), without altering total or esterified cholesterol ³²⁵. Fatty acid fluidity might also affect lipoprotein packing and surface protein conformation ¹²³, as well as lipid droplet hydrolysis and cholesterol efflux ^100,224,326.

Lipid microdomains are directly implicated in vascular function and atherogenesis. For instance, LDL and hypercholesterolemia inhibit endothelial nitric oxide synthase (eNOS) via translocation to caveolae rafts (reviewed in ⁵²). Similarly, in human atherosclerotic plaques the oxLDL receptor LOX-1 is associated with caveolae and dissociated by statins or MβCD (which extracts membrane cholesterol), thereby abrogating oxLDL-induced apoptosis ³²⁷. As above, LDL can also deliver ceramide to endothelial cells ¹⁸³, where endogenous ceramide promotes the uptake and retention of oxLDL via regulation of transcytosis-related and raft-associated proteins, including LOX-1 ¹⁸⁵. Further, conditions of hypercholesterolemia and 7-ketocholesterol induce endothelial A-SMase/ceramide-dependent membrane raft redox signalling platforms linked to NLRP3 activation ³²⁸. Notably, electronegative LDL also possesses intrinsic SMase activity associated with apoB₁₀₀ serine O-glycosylation ³²⁹; this may be outward-facing so as to engage plasma membrane sphingomyelin, generating ceramide-based microdomains and endocytic vesicles ^330,331. Moreover, arterial SMase can hydrolyse sphingomyelin within lipoproteins themselves generating ceramide-rich domains, which may act as nonpolar spots promoting aggregation via hydrophobic interaction ⁴⁹, as well as displacement and release of cholesterol to neighbouring vesicles ³³². Atherosclerotic plaques were also reported to contain membranes enriched in free cholesterol and crystalline domains ⁵². In preclinical studies plaque crystals co-associated with cholesterol microdomains ³³³, which can be shed from macrophage membranes ³³⁴. Rapid loading of macrophages via phagocytosis of large lipid droplets induces lysosomal free cholesterol and extracellular crystals ⁴⁷; inhibition of esterification also induces crystals and cytotoxicity offset by extracellular acceptors (i.e. apoA-I/E) mediating efflux of cholesterol from the plasma membrane ^335,336. Further, lipid oxidation induces crystalline domains in model membranes under conditions of hyperglycaemia, which can be inhibited by n-3 PUFAs (esp. EPA) ³³⁷. More specifically, smooth muscle cells grown in the presence of 7-ketocholesterol produced extracellular crystals via formation of distinct membrane microdomains due to reduced intercalation with phospholipids ³³⁸.

Regarding immuno-pathogenesis, in human cohorts LDL-c correlated a haematopoietic monocyte skewing (vs. granulocytes) in blood ⁷⁸ and proinflammatory macrophage phenotype in adipose ³³⁹, which were suppressed by statins. One potential pathway involves CD131, the common β subunit of GM-CSF and IL-3 receptors. Accordingly, in LDLR^–/– mice administration of lipid-free apoA-I reduced aortic cholesterol and macrophage deposition, as well as systemic CD131⁺ immune cells and their CE content, while in vitro LDL and apoA-I oppositely regulated monocyte membrane cholesterol shifting CD131 between raft and non-raft fractions, respectively ³⁴⁰. In addition, in an RCT on obese individuals, dual lipid-lowering therapy for 6 weeks markedly lowered apoB and lipids (e.g. LDL-c 141–73mg/dl), as well as fasting and cream/SFA-induced LPS and immune cell activation; postprandial PBMC TLR2/4 expression was even below baseline (i.e. Fig. 3G/H) ²⁹⁷. Of relevance here, in LPS-stimulated macrophages TLR4 activation requires cholesterol biosynthesis (via FASN) to enter lipid rafts ³⁴¹, while ABCA1-dependent cholesterol efflux suppresses raft-associated TLR/inflammatory signalling in macrophages ^342,343 and endothelial cells ³⁴⁴.

Shortly after the discovery of TLR4 as the LPS receptor it became apparent that fatty acids could also modulate TLR signalling ³⁴⁵. In particular, at higher levels than bacterial ligands, free SFAs can also induce TLR4 and 2 signalling (esp. lauric and palmitic acid) via NOX/ROS and cholesterol-dependant rafts ^346,347, and potentiate that by TLR ligands, all of which is inhibited by UFAs (esp. DHA) ^345,348. Paralleling this, bacterial LPS and lipopeptides are acylated with chains of SFAs which are required for TLR4 and TLR2 signalling, respectively. For instance, in E. coli LPS the lipid A region is typically hexa-acylated with C14/12 SFAs ³⁴⁹, while hypo-acylation or incorporation of UFAs result in antagonist activity ^345,348; similarly, total gut LPS silences TLR signalling due to hypo-acylated lipid A in Bacteroidales ²⁴⁸. Cellular sensitivity to low levels of LPS is supported by initial binding to surface CD14/CD36, which facilitates transfer to the TLR4–MD2 complex ³⁵⁰, wherein its saturated acyl chains interact with MD2 lipid domains inducing TLR4 dimerization ³⁴⁸. As a corollary, free fatty acids (SFAs and UFAs) may also bind within the hydrophobic pocket of MD2 to directly modulate TLR4 signalling ^348,351,352, while other evidence suggests palmitate acts indirectly via lipid metabolism and ER stress ³⁵³. As above, lowering the dietary palmitate/oleate ratio can lower PBMC LPS sensitivity, while principle component analysis implicated corresponding changes to tissue lipids in the mechanistic pathway ³⁰¹. In line with this, in mice systemic inhibition of the ER-associated enzyme SCD1, which mediates endogenous desaturation of SFAs to MUFAs, induced macrophage TLR4 hypersensitivity ³⁵⁴. Since plasma membrane rafts are rich in polar lipids with saturated acyl chains and can be modulated by SCD1 ³⁵⁴ and n-3 PUFAs ³⁴⁸, this may contribute to general effects. LPS and oxidative stress-induced raft–TLR4 complex formation also requires A-SMase-derived ceramide ^355,356 (which has structural similarities to lipid A ³⁵⁷), while palmitate augments LPS inflammatory responses via SMase and de novo ceramide synthesis ^358–361. Free SFAs (i.e. palmitic and stearic acid) can also activate macrophage NLRP3 inflammasomes via flux into phosphatidylcholine and ER stress ³⁶², and even crystallisation ³⁶³, which are offset by UFAs. Consequently, SFAs may stimulate and sensitise innate immune signalling in various ways and have been suggested to mediate LPS-associated postprandial inflammation ³⁶⁴.

Lipid rafts are also involved in endocytosis ³¹⁶ and represent a common entry point for many viral, bacterial and fungal pathogens ^365–367. For instance, in the colon butyrate may inhibit enteric pathogen invasion via depletion of cholesterol and increased membrane fluidity ³⁶⁸. Similarly, in porcine ileum samples SFA-induced LPS permeability was abrogated by MβCD, implicating lipid rafts ²⁹⁴. Another study using oleate and taurocholate (which is especially induced by SFAs ²⁹⁰) further implicated a raft/CD36-dependent pathway ³⁰⁷. Of additional interest, intestinal enterocytes were reported to phagocytose E. coli/LPS via TLR4, while in mice TLR4 deficiency prevented bacterial translocation in response to injury ³⁶⁹, and induction of faecal/plasma LPS by a high fat/lard diet ³⁷⁰. The differential effects of fatty acids on TLR4 signalling ³⁴⁵ also parallels those on postprandial LPS ²⁹⁴.

Ecology

Evidence of atherosclerosis has been reported in ancient humans spanning 4000 years and from diverse locations suggesting a basic predisposition ³⁷¹; patterns of systemic vascular calcification were even similar between ancient and modern Egyptians, appearing in aorta–iliac beds almost a decade prior to event-related coronary and carotid beds ³³. These populations may have been exposed to various enduring risk factors, including diet, smoke and infections, although this remains speculative ³⁷². However, there are some notable examples of extant pre-industrial people with divergent health outcomes, suggesting post-industrial changes are also important ³⁷³. Foremost, the Tsimane forager-farmers of Bolivia are a tropical subsistence population with a high infectious/inflammatory burden, yet some of the lowest ever reported coronary artery calcium (CAC) scores throughout life ³⁷⁴; as well as atrial fibrillation ³⁷⁵, age-related brain atrophy ³⁷⁶ and dementia ³⁷⁷.

Lipids likely play a fundamental role in our susceptibility to CVD. Indeed many other mammals are relatively resistant to atherosclerosis and exhibit low apoB-lipoproteins so their use as experimental models requires induction of hyperlipidemia via manipulation of genes and diet ⁵⁴; although important differences may still remain, such as ACAT2-dependent lipid sensitivity ^42,113. Experimental atherosclerosis also requires NLRP3, which links microbial and/or sterile cell stress to innate immunity ⁷⁵. Today many human infections are associated with atherosclerosis and the acute-phase response encompasses mutual changes supporting immunometabolism and defence reminiscent of those implicated in CVD ^378,379. For instance, infections/inflammation induce macrophage aerobic glycolysis and accumulation of lipid droplets with antimicrobial activity ^380,381, while also modulating systemic insulin sensitivity (i.e. glucose metabolism) ^382,383, lipid metabolism (e.g. lipolysis, cholesterol and sphingolipids) and lipoprotein modifications (e.g. oxLDL) ³⁷⁹. Induction of vascular retention and LDL oxidation might even support bacterial sequestration (e.g. LPS­–TLR4 ^249,280 and M. tuberculosis ⁴⁶) and phagocyte clearance (e.g. LPS ^281,282). These mechanisms could therefore support acute survival, while persistent stimulation exacerbates vascular disease hastening late-life mortality ³⁷⁹, as a form of antagonistic pleiotropy. Indeed while CVD is currently the leading cause of death, ancestrally it was likely infections and injury ³⁷³, underscoring the evolutionary priority.

Many bacterial pathogens are Gram-negative with a distinct outer membrane coated in LPS, which confers barrier function and protection from antibiotics ³⁸⁴. The specific composition of LPS varies between bacteria and is modulated by environmental factors ³⁸⁴. In pathogenic bacteria the lipid A region is typically hexa-acylated with SFAs ³⁴⁹, which may impart a gel-like state and low permeability, while also mediating activation of innate immunity in mammalian cells via L_o microdomains, which are similarly enriched in SFAs ³⁸⁵. As above, SFA-rich diets may act on gut and liver to increase circulating immunogenic lipids (e.g. cholesterol, ceramides and LPS) which converge on membrane microdomains. Further, in healthy adults SFA intake and SCD (aka. ∆9-desaturase) activity also correlated the kynurenine/tryptophan ratio (a surrogate of IFNg/Th1 activity), which itself correlated CRP ³⁸⁶. In mice SFA-rich diets (i.e. C12/16:0) exacerbated central autoimmunity by increasing Th1/17 activity via the small intestine ^387,388. Th17 cells express particularly high levels of TLR4, and LPS directly induces Th17 differentiation in vitro ³⁸⁹. Accordingly, Th17 lymphocytes are part of type-3 immunity which mediates antimicrobial responses to extracellular pathogens (e.g. Gram-negative bacteria), although when dysregulated also autoimmunity ³⁹⁰. In addition, in Rag1^–/– mice lacking adaptive immunity, a SFA-based ketogenic formula enhanced clearance of C. albicans, while simultaneously conferring susceptibility to endotoxemia, which involved palmitate and ceramide, persisted for 7 days post-exposure, and was reversible with oleate ³⁹¹. This was consistent with SFA induction of innate immune memory (i.e. ‘trained immunity’), itself another double-edged sword ³⁹². As above, plasma cholesterol may similarly stimulate immune cells, and while treatment of human hypercholesterolemia with statins normalised monocyte skewing and lipid droplets, an activated phenotype persisted, again implicating trained immunity ⁷⁸. Dietary cholesterol may also affect the pathophysiology of infectious and autoimmune disease (reviewed in ³⁹³).

The ability of exogenous lipids to differentially regulate endogenous lipids and physiology may arise from several key factors. Foremost, the dependence of membrane biophysics and cell signalling on specific fatty acids means lipid saturation must be tightly regulated; in particular, excess long-chain SFAs can induce ER stress/inflammasome pathways offset by UFAs ^323,362,363. Accordingly, ER-associated SCD1 mediates endogenous desaturation and limits experimental atherosclerosis and macrophage TLR4 hypersensitivity, potentially via suppression of L_o microdomains, but at the cost of obesity-related metabolic disease ³⁵⁴. However, mammalian fatty acid metabolism is imperfect and especially limited at PUFA synthesis, underlying the essential fatty acids (i.e. C18:2/3) and modulation by diet. Similar issues pertain to cholesterol, which is also fundamental to membrane biophysics, but in excess can precipitate as crystals and exert toxicity. While most cells can synthesise cholesterol its catabolism is limited in extrahepatic tissues to side chain oxidation (via sterol 27/24-hydroxylases) and steroidogenesis (in hormonal glands), with ultimate conversion to bile acids occurring in the liver. Therefore excess cholesterol must be esterified for storage or effluxed (via HDL, RBCs or albumin ^41,219) for transport to the liver and intestine for biliary and direct excretion, respectively. Fatty acid and cholesterol metabolism directly converge on the formation of cholesteryl esters, which have a preference for UFAs (i.e. ACAT ^112,116 and LCAT ^119–121), suggesting their availability may influence cholesterol turnover and therein processes of uptake/efflux, transport and crystallisation. Further, the role of lipids and lipoproteins in immunity may superimpose another layer of regulation mostly concerned with acute survival ³⁷⁹. In particular, LPS–TLR4 signalling induces macrophage cholesterol synthesis ³⁴¹ and inhibits systemic reverse cholesterol transport ²⁸³, presumably creating a positive sterol balance.

Consequently, these metabolic constraints and connections create a susceptibility to different foods and associated environments, which themselves may confer adaptive or maladaptive effects on short-term fitness or long-term health, in concert with genomic variation. How might this play out through our evolution? The other great apes from which we diverged are highly plant-based (i.e. fruits and leaves), after which our diet became increasingly diverse and animal-based with our spread to colder environments and eventually pastoralism ³⁹⁴. Initially the hepatic LDL shunt pathway may have evolved to favour cholesterol conservation ⁴³, while an increase in dietary cholesterol and fats from animals (i.e. land and marine) and plants (esp. nuts/seeds) may have further modulated plasma cholesterol in relation to the SFA/PUFA ratio ^{89,90,94,112,114}. Notably, since meat can deteriorate quickly, this might have also increased exposure to pathogens, consistent with our relatively low stomach acid pH (i.e. similar to scavengers) ³⁹⁴ and animal food aversion during early pregnancy (i.e. morning sickness), a relatively immune-suppressed period ^395,396. The advent of cooking would support sterilisation and may be reinforced by the appealing sensory qualities of advanced glycation end products (AGEs) ³⁹⁷, albeit at the potential cost of cardiometabolic dysregulation, as seen in modern humans ³⁹⁸. Despite these factors native populations often exhibit relative cardiometabolic health ^373,399. For instance, our genus and species emerged in Africa, where remaining hunter-gatherer exemplars such as the Hadza of Northern Tanzania maintain low body weight, blood pressure and plasma lipids throughout life ³⁷³, with a low fat intake (i.e. median ~18% kcals) from plants and lean meats ³⁹⁴. Similarly, Tsimane vascular health is accompanied by a low LDL-c (esp. till 2011) ³⁷⁴ and fat/SFA intake (i.e. men: 15.1/3.7% kcals, respectively) from a plant-dominant diet with moderate fish/meat ⁴⁰⁰. Furthermore, in this energy-limited and pathogenically diverse context, the ancestral APOE4 allele is actually associated with better cognition in those infected with parasites ⁴⁰¹, and slightly increased lipids (i.e. cholesterol +2.8% and oxLDL +3.9%) and lower innate immune markers (e.g. CRP –21.6%), which were inversely associated in those with a lower BMI only ⁴⁰², suggesting it may not have the same deleterious effects as in post-industrial populations, but instead support cognition and immunity. Notably, in a modern UK population, carriers of the APOE4 allele (vs. E3/E3 and E2) achieved the lowest plasma cholesterol and apoB when replacing SFAs with low GI carbohydrates (vs. high GI and MUFAs) ⁹⁸.

Like many isolated native populations the Tsimane are now in a state of nutritional transition as they increasingly interface market towns, with corresponding changes to cardiometabolic health of increasing body fat ⁴⁰⁰ and plasma lipids ³⁷⁴ (see Table S6). Earlier clinical studies on the Tarahumara Indians of Mexico showed their similarly low plasma cholesterol increases rapidly in response to dietary cholesterol ⁴⁰³ and an ‘affluent’ diet ⁴⁰⁴. In industrialised societies major SFA sources are now grain-fed meats (which are richer in fat and lower in n-3 PUFAs than grass-fed ⁴⁰⁵) and concentrated/added fats from animals/dairy and tropical plants (as used in SFA trials herein), in the context of a diet high in ultra-processed foods/calories (and plant oils/n-6 PUFAs ²⁷) and low in micro-/phytonutrients, implicating evolutionary mismatch in SFA-associated diseases ³⁹⁹. Indeed many nutritional and physiological factors may modulate the effects of dietary SFAs today (and serve as study confounders). For instance, SFA-induced elevations in plasma cholesterol may depend upon intake of dietary cholesterol ^89,90, plant-based PUFAs ^112,114 and associated phytosterols ^130,131. SFA-induced postprandial inflammation may especially occur in obesity ^297,298,406, but be blunted by lipid-lowering therapy ²⁹⁷ or co-ingestion of phytochemicals (e.g. polyphenols ⁴⁰⁷, spices ⁴⁰⁸ and fibre ⁴⁰⁹), which can also accompany UFAs (e.g. olive oil and nuts). Of UFAs, cellular levels of long-chain (marine) n-3 PUFAs are particularly responsive to diet (at the expense of n-6 PUFAs) ⁴¹⁰ and exhibit robust anti-LPS/TLR effects ^{290,293,294,345,348} (not without potential for excess ^411,412), implicating omega-3 status. SFA-induced liver fat may be promoted by poor metabolic health ⁴¹³, overfeeding ^{159,161–163} and excess fructose ³⁰⁹. Conversely, low carbohydrate diets increase fat oxidation and may mitigate the differential effects of SFAs (vs. UFAs) on insulin sensitivity and inflammation, but not cholesterol, SCD and ketones ^82–86. In particular, ketosis may have inherent anti-inflammatory effects ⁴¹⁴, and a 3-day isocaloric ketogenic diet suppressed LPS/palmitate-induced inflammasome activation in macrophages ex vivo ⁴¹⁵.

While many factors may contribute to atherogenesis, as a condition of arterial lipid accumulation, a lipid-threshold may ultimately govern its progression ^416,417; indeed atherosclerosis rarely occurs in mammals and humans with an LDL-c <80mg/dl ^46,317. Accordingly, in the PESA study of a healthy middle-aged Spanish cohort ³¹, systemic subclinical atherosclerosis became increasingly rare and undetectable at low LDL-c values, albeit with an increasingly small sample size ³². Moreover, decades of research on experimental atherosclerosis shows that most aspects of advanced plaques can regress/reverse, including necrotic and crystalline material, in association with dramatic lipid lowering and improved HDL function ^74,418,419. Similarly, in humans athero-regression can be induced with intensive lifestyle changes ⁴²⁰ (with <SFAs ^80,177) and/or lipid-lowering drugs ⁴²¹ (with LDL-c <80mg/dl ^422,423); and some extreme cases have been reported ^424,425. In fact when considering other mammals, newborn humans and native populations, these low cholesterol levels may even be physiologically normal ^317,426; in which case athero-regression could simply reflect a return to the natural homeostatic state. So despite its ubiquity, perhaps atherosclerotic CVD is not inevitable, at least not without a chronic deviation from physiological homeostasis, as a result of relatively rare mutations (i.e. familial hypercholesterolemia), or more generally, a maladaptive environment.

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