From saturated fat to arterial plaque: cholesterol and beyond

This article is a work in progress and regularly updated as I study this topic.

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 inconsistency 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 SFAs (mainly from animals) with unsaturated fatty acids (UFAs; from plants and fish) or complex carbohydrates (from whole grains) 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 early observational research many randomised controlled trials (RCTs) were also conducted, and which have also been subject to contemporary meta-analysis with somewhat inconsistent results ^14,15. Unfortunately these trials are mostly old and heterogeneous, with some confounded by trans fats ¹⁶, making any pooled analysis highly sensitive to the inclusion/exclusion criteria. Nonetheless, there is often a trend for ¹⁴, or significant benefit ^15,16. For instance, in the Cochrane meta-analysis of 15 trials, lowering SFAs (typically from animals) 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) ¹⁵. And focusing in further, in a more stringent analysis restricted to 4 core trials with PUFA replacement (mostly via seed oils, particularly soybean) the results were stronger (RR=0.71, 95% CI 0.62–0.81) ¹⁶. Supporting this favourable effect on hard endpoints, a diverse literature of shorter RCTs show replacement of SFAs can favourably modulate various biomarkers of risk and pathophysiology within blood lipids ^17,18, immuno-metabolic health ^19–23 and endothelial/platelet activity ^24–26.

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 ^27–30. Indeed as well as methodological difficulties with the observational and interventional data 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. Moreover, mechanistic 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 suggested by qualitative comparisons with UFAs in particular. To this end searches were performed with PubMed and Google Scholar, prioritising human studies and congruent preclinical studies. Relevant pathways affected by dietary fats were grouped under 4 core themes/sections: cholesterol, metabolic, oxidation and microbiome; while additional sections explore microdomains as a common site of action, and the ecological basis of all the above.

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. Indeed from a functional perspective, early stages involve largely superficial intimal thickening and substantial plaque accommodation by positive remodelling ³¹, beyond which growing plaque increasingly protrudes the lumen and calcifies, constraining blood flow and vasodilation ³², with impending potential for rupture, thrombosis and infarction (e.g. heart attack or stroke). As such, symptoms are not realised until advanced or end-stage disease. Further, as clinical assessment focuses on event-related beds (e.g. coronaries), its full extent is also rarely appreciated; systemic in vivo imaging of general populations suggests subclinical plaque (i.e. stenosis or calcification) may be present in around half of asymptomatic individuals by midlife (e.g. US ³³, Scotland ^34,35, Spain ^36,37 and Egypt ³⁸), affecting many arteries (most frequently aorta–iliac–femoral beds) and correlating brain hypometabolism ³⁶. From a histological perspective, early lesions (AHA types I–III) not yet visible on angiography are characterised by intimal accumulation of lipids and leukocytes (esp. macrophages), while advanced lesions develop a well-defined lipid core (type IV) and fibrous cap (type V) ³⁹, the exact composition and morphology of which may vary by arterial region and risk factors ⁴⁰. The lipids are largely cholesterol-related: i.e. cholesteryl esters (CEs) within lipid droplets of foam cells and extracellular deposits, as well as free and crystallised cholesterol—a hallmark of the atheroma core ⁴¹; while the simultaneous presence of apolipoprotein B (apoB) implicates plasma lipoproteins as a source ^41–44.

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 of triglycerides (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 and esterifies cholesterol from the periphery maturing into high-density lipoprotein (HDL), which transfers 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 (typically >90%). Ultimately, plasma cholesterol is largely cleared by the liver (i.e. reverse cholesterol transport ⁴⁵); indeed unlike triglycerides (and phospholipids), cholesterol cannot be fully catabolised by extrahepatic cells. Once in the liver cholesterol can be recycled or excreted via bile, fates which may depend upon the form of delivery: in humans the bulk of HDL-CEs (~70%) are transferred to VLDL/LDL before hepatic uptake, however, uptake of free cholesterol, particularly from HDL, may favour biliary excretion ^46,47. Of note, some preclinical studies also suggest hepatic channelling of cholesterol from LDL to plasma ⁴⁸ and HDL to bile ⁴⁹; although LDL can also mediate macrophage efflux ⁵⁰ and the LDL receptor (LDLR) contributes substantially to faecal excretion in mice (which are naturally CETP deficient) ⁵¹.

Lipoproteins can readily traverse the arterial wall where they may support normal cellular lipid metabolism ^52,53, but accumulate during atherogenesis ^54–56. Specifically, in early human coronary lesions initial deposition of extracellular lipid and apoB occurred deep in intima (above internal elastic lamina) and prior to macrophage infiltration ⁴⁴. Susceptible sites (e.g. bends and bifurcations) are typically exposed to turbulent blood flow and low shear stress, and may exhibit increased lipoprotein permeability and/or retention ^54,57. However, contrary to a passive paracellular influx, free-LDL levels in normal arterial interstitial fluid may already be higher than plasma ⁵⁸, and lipoproteins cross the endothelium via transcytosis (i.e. active transport) ^53,59, which is upregulated in human plaque and murine atherogenesis via increased expression of scavenger receptor class B type 1 (SR-B1) ⁶⁰. Further, plaque-prone regions exhibit diffuse intimal thickening, with increased content of smooth muscle cells and extracellular matrix ^44,61. Here apoB-lipoprotein binding to proteoglycans (via electrostatic interaction) and LpL (acting as a bridge) may promote retention ^44,54,55, while exposure to various enzymes and oxidants promote modifications, ultimately resulting in aggregation, fusion and cholesterol crystallisation ^43,62,63 (as per the ‘response-to-retention’ hypothesis). Another key event is the formation of foam cells, which may arise mainly from smooth muscle and myeloid cells (i.e. monocytes and macrophages) via ingestion of lipoproteins ^64,65. 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 become a site of crystallisation, inflammation and cytotoxicity ^56,66,67, which may underlie development of the necrotic lipid-rich core ⁴¹. In this regard, lipoprotein aggregates isolated from human plaques induce accelerated macrophage uptake, greater cholesterol esterification ⁶⁸ and inflammasome activation ⁴³, while recovered apoA-I/HDL is lipid-poor and pro-inflammatory, suggesting low acceptor/efflux activity ⁶⁹. Reverse cholesterol transport from tissues may also be mediated by lymph ^53,58,70 and plaque progression is accompanied by expansion of adventitial lymphatics, which in murine models can modulate atherogenesis ⁷¹. Beyond the arterial wall, atherosclerosis is also associated with increased haematopoiesis ⁷²; even in apparently healthy people with subclinical atherosclerosis bone marrow activity (i.e. labelled glucose uptake) correlated blood immune-inflammatory markers and arterial activity (a surrogate of macrophage activity), suggesting early links to atherogenesis ⁷³.

Cholesterol

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) and is now supported by meta-analyses of many prospective cohorts ⁷⁴. Further, studies explicitly on low risk populations also find LDL-C is linearly and independently associated with subclinical atherosclerosis and CVD mortality ^37,75–77; even when including markers of LDL subspecies such as HbA1c (i.e. glycation), oxidised LDL (oxLDL) and lipoprotein(a) ³⁷. Of note, in older cohorts associations with all-cause mortality may be inverted by malnutrition ⁷⁸. 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) ^74,79. However, CVD risk can track better with the cholesterol content of other apoB-lipoproteins (e.g. VLDL, remnants and non-HDL) and indeed apoB itself ^80,81, any of which can be discordant from LDL-C, especially in situations of elevated triglycerides/VLDL and a preponderance of small-dense LDL particles (i.e. pattern B), as in metabolic disorders discussed later. Such small (vs. large) LDL particle profiles have also been associated with greater risk ⁸², although this disappears when controlling for particle count and other confounders ^83,84; indeed small-dense LDL exhibits properties which may favour retention ⁸⁵, albeit while carrying a smaller cholesterol load ⁸⁰. Further, recent Mendelian studies also suggest the risk from apoB is mediated by non-HDL-C ^86,87, 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 risk better than HDL-C, suggesting HDL function is more important ⁴⁵.

The atherogenicity of apoB-lipoproteins and cholesterol is supported by preclinical studies. Indeed since the early 1900s, and the work of Anichkov, it was known feeding some animals diets high in cholesterol can induce atherosclerosis ⁸⁸. Nowadays mouse models are ubiquitous and typically employ genetic manipulation (e.g. LDLR or APOE deficiency) and a lipid-rich diet to increase apoB-lipoproteins and atherosclerosis ⁸⁹. Mechanistically, 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 important 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 ⁹⁰ and selective CE uptake ⁵⁰; as well as proteoglycan secretion ⁹¹. LDL transcytosis through endothelium is also dose-dependent ⁵², and high or prolonged exposures can induce endothelial dysfunctions (e.g. adhesiveness ⁹², nitric oxide ⁹³, permeability ^94,95 and senescence ⁹⁶), which may involve cholesterol itself, as discussed later (and reviewed in ^59,66,97). In particular, native LDL treatment of human endothelial cells for several days induces lipid droplets and cholesterol crystals, which are reduced by cAMP stimulation ⁹⁸. Cholesterol crystals can impair endothelial function (e.g. vasodilation, leukocyte barrier and cell survival) ^98,99, while activating endothelial and macrophage NLRP3 inflammasomes, which release cytokines recruiting immune cells ¹⁰⁰ and inducing LDL transcytosis (via LDLR) ¹⁰¹. In hyperlipidemic mice on a western (sugar/SFA-rich) diet cholesterol crystals form within a week (i.e. before macrophage infiltration and neointima formation), while stimulation of cAMP (in inflamed endothelium) ⁹⁸ or deficiency of NLRP3 (in bone marrow) can suppress early atherosclerosis ¹⁰⁰. Thus native LDL is not innocuous, but may be decoupled from atherogenesis via modulation of endothelial and immune activity, the physiological states of which may determine individual susceptibility. Counterplay with HDL/efflux may also determine thresholds in vivo and seems rarely tested in vitro ^50,102.

Regarding diet, in the Cochrane analysis of SFA-replacing 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 ¹⁵. 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) ¹⁰³. Indeed isocaloric replacement of complex carbohydrates with typical dietary SFAs (i.e. C12–16:0) raises total/LDL-cholesterol, whereas plant-based PUFAs (i.e. C18:2/3) lower it, as established via 100s of metabolic ward studies ¹⁷ and formularised since the 1950s ¹⁰⁴. This SFA/UFA dichotomy extends to low carbohydrate/ketogenic diets ^105–109. Sensitivity is also maintained long-term; in the LA veterans trial a PUFA-rich diet lowered serum cholesterol up to 8 years and with return to a conventional diet it reverted within 1–2 weeks ¹¹⁰. The cholesterol-raising effect of SFAs may be accentuated by dietary cholesterol ^111,112 and attenuated in the case of cheese (vs. butter) ¹¹³. Alongside cholesterol, SFA-rich diets also increase apoB ^26,113–117 (for meta-regression see ¹⁸); more specifically, dairy fat/SFAs can increase all VLDL–LDL particles (vs. seed oils/n-6 PUFAs) ^114,118, large LDL (vs. MUFAs) ¹¹⁶ or medium–small LDL particles (vs. MUFAs in people with pattern B) ¹¹⁷. Whereas replacing SFAs with MUFAs or PUFAs did not significantly affect lipoprotein(a) in a recent meta-analysis of RCTs ¹¹⁹. Individual sensitivity to SFAs is also heterogeneous ²⁶ and depends on genotype, most notably APOE variants ^120,121; for instance, in the UK RISCK study (n=389) carriage of an E4 allele (vs. wildtype E3/E3) increased the cholesterol/apoB-lowering effect of replacing SFAs with low GI carbohydrates, while reducing that of MUFAs ¹²⁰.

The atherogenic effect of SFAs is also evident in animal models such as non-human primates ¹⁶. For instance, in African green monkeys fed cholesterol-containing diets with 35% fat for 5 years, safflower oil/n-6 PUFAs (vs. palm oil/SFAs and safflower oil/MUFAs) reduced atherosclerosis, which correlated LDL-C and particle weight/size ¹²². Across various genetic mouse models replacement of SFA-rich diets with seed oils/n-6 PUFAs can also reduce hyperlipidemia and atherosclerosis (in spite of increased inflammation) ^123–126. Further, 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 ¹²⁷; whereas in LDLR^–/– mice incremental replacement with fish oil/n-3 PUFAs reduced hyperlipidemia (esp. cholesterol), arterial macrophages/LpL and aortic lesions ¹²⁸. In APOE^–/–mice hypercholesterolemic diets also rapidly (within days) induce foamy-inflammatory monocytes which infiltrate nascent lesions ¹²⁹; in LDLR^–/– mice this was reduced by replacing dairy fat/SFAs with plant-based UFAs (i.e. extra-virgin olive oil and nuts) ¹³⁰. Note 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 ^122,126. In mice this requires ACAT2 (aka. SOAT2) ¹²⁶ which synthesises oleate-rich CEs for apoB-lipoproteins ¹³¹ and mediates LDL proteoglycan binding ¹³² and aggregation ¹³³; contrasting the more favourable effects in humans of MUFA-rich diets on LDL size ^116,117 and binding ^132,134. Importantly, animals may be fed higher cholesterol ^132,135, express higher ACAT2 (e.g. monkeys and rats) ^46,136 or have LDLR/apoE knockout (e.g. mice) ⁸⁸, any of which might increase MUFA sensitivity; while effects in humans may depend on food source ^7,8, olive oil quality ¹³⁷ and APOE variants ¹²⁰.

How does fat saturation modulate plasma cholesterol? In human trials SFA-rich diets decrease LDL catabolism ¹³⁸ and PBMC LDLR expression ^26,114,115, which inversely correlates serum apoB/LDL-C ¹¹⁵, suggesting decreased tissue uptake. In animal models this depends upon dietary cholesterol ^135,139,140. Along this line, early hamster 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 ¹³⁵. However, in monkeys and rodents MUFAs stimulate the greatest hepatic CE synthesis/secretion ^141,142 and LDLR expression ¹³⁵; and in LDLR^–/– mice MUFAs and SFAs elevate plasma cholesterol over PUFAs ^123,125, which was largely abrogated by ACAT2 deletion ¹²⁶. Thus ACAT has specificity for oleate, yet plasma lipids are lowest with linoleate. Moreover, 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 ⁴⁶. Rather human plasma CEs are typically rich in n-6 PUFAs (i.e. C18:2 > C18:1 > C16:0), in contrast to triglycerides and phospholipids ¹⁰⁶, and consistent with the specificity of LCAT, which mediates cholesterol esterification in plasma lipoproteins via transfer of a fatty acid from phosphatidylcholine, favouring sn-2 position C18:2 ^143,144. Dietary fats can modulate plasma phospholipid composition, and in some human and rodent studies also endogenous LCAT activity (i.e. n-6 PUFAs > MUFAs > SFAs) ^145–147, although other findings are less consistent ^145,148,149. In addition, overexpression of human LCAT in animals such as transgenic rabbits (which also naturally express CETP) typically elevates HDL-C, and may lower apoB/LDL-C via the LDLR ¹⁵⁰, although large increases in enzyme quantity (vs. substrate modulation) may have different effects. Regardless, in humans most HDL-CEs are transferred to VLDL/LDL and fatty acid composition may affect cell binding. For instance, in men with hypercholesterolemia a 6-week walnut/PUFA-rich diet (vs. olive oil/MUFAs) increased LDL n-6/n-3 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 r²=0.41) ¹⁵¹. Besides LDL, PUFAs (vs. SFAs) may lower HDL-C in humans via decreased apoA-I production ¹⁵², whereas in other animals via increased HDL clearance ^148,153.

Does fat saturation affect systemic sterol balance? Early human studies varied in their methods and results, but PUFAs (vs. SFAs) more often increased sterol excretion (i.e. faecal neutral sterols and/or bile acids) ^154,155 and cholesterol biosynthesis (i.e. deuterium incorporation) ¹⁵⁶, which seem highly related ¹⁵⁷. The former may particularly occur at high (vs. low) cholesterol intake and under non-steady state in normal adults, but less so in familial hyperlipidemia, despite still lowering plasma cholesterol; thus tissue redistribution has been suggested ¹⁵⁴, as seen in guinea pigs (on low cholesterol diets for 6–7 weeks) ¹⁵⁸. The sterol balance technique has also been adapted to ileostomy subjects where isocaloric fat substitution with oils rich in PUFAs or MUFAs acutely increased net sterol excretion (within 2–4 days) ¹⁵⁹, which may be partly attributable to their phytosterol content ¹⁶⁰ (as controlled for in several earlier studies ^154,155,161). Some studies also implicate upstream changes to reverse cholesterol transport. For instance, healthy adults (n=122) with higher insulin responses or SFA intake (>10% kcal) had lower ABCA1-dependent efflux to HDL (independent of HDL-C); tested in mice an obesogenic diet rich in palm oil/SFAs or sunflower oil/MUFAs lowered ABCA1-dependent efflux, but only the former lowered liver­–faeces cholesterol transport (in relation to weight gain) ¹⁶². Further, replacing butter/SFAs with seed oils/n-6 PUFAs (without controlling for dietary sterols) for 8 weeks induced serum bile acids as well as PBMC transcripts related to efflux (LXRα and ABCG1) and influx (LDLR) (also replicated in ²⁶); in multivariate analysis (incl. lipids, metabolites and gene expression) the most important explanatory variable was LXRα ¹¹⁴. After just 3 days this intervention also induced faecal Bifidobacteria and Lachnospiraceae, the latter negatively correlating total cholesterol (r=­–0.511); as a potential mechanism the authors invoked other evidence suggesting bacterial conversion of faecal cholesterol to coprostanol may prevent reabsorption ¹⁶³. In summary, dietary UFAs (vs. SFAs) may lower plasma cholesterol largely by increasing LDL uptake (by liver and elsewhere ¹⁵⁸), and concomitant with sterol efflux/excretion ^114,140. Whether such observations in PBMCs ^26,114 will extend to immune cells within arterial lesions and support reverse transport is explored more later.

Metabolic

Recent decades have seen an epidemic of obesity and overweight-related metabolic disorders, including non-alcoholic fatty liver disease (NAFLD), metabolic syndrome (MetS) and type-2 diabetes (T2D), which are strongly associated with one another and CVD. In people with subclinical atherosclerosis bone marrow activation was also associated with MetS and its components (but not LDL-C), even in those with lower systemic inflammation (i.e. below median CRP) ⁷³. Such metabolic disorders have shifted the typical lipid profile toward increased serum triglycerides and low HDL-C ¹⁶⁴, with attendant small-dense LDL (aka. ‘atherogenic dyslipidemia’) ⁸⁵. Linking this profile, high hepatic triglyceride status drives overproduction of VLDL₁ (enriched in apoC-III), triglyceride transfer to LDL (via CETP) and lipolysis to small-dense LDL (via hepatic lipase); and a similar process may lower HDL-C ^85,165. Importantly, triglyceride-rich lipoproteins (TRLs) also constitute non-LDL/HDL-associated ‘remnant cholesterol’ ¹⁶⁶, but may play a potent causal role in atherosclerosis beyond their cholesterol content (and LDL) as suggested by Mendelian ⁸⁷ and preclinical studies ¹⁶⁷.

Regarding diet, in meta-regression of RCTs isocaloric replacement of carbohydrates with SFAs can lower fasting triglycerides, but UFAs more so (i.e. C18-PUFAs > MUFAs > SFAs) ^16,18. Further, in overweight people with a small LDL particle profile (i.e. pattern B), a dairy/SFA-rich diet (vs. MUFAs) for 3 weeks non-significantly increased triglycerides (p=0.06), but significantly increased apoB, medium–small LDL particles and hepatic lipase activity, and CETP activity independently correlated medium and small LDL ¹¹⁷. While effects on postprandial lipemia seem equivocal (reviewed in ¹⁶⁸), SFA-rich meals (i.e. palm oil and cocoa butter vs. various UFAs) induced apoE expression on TRLs; tested in vitro this increased hepatocyte LDLR binding causing competitive inhibition of LDL uptake ¹⁶⁹, and more so in those with an APOE4 allele ¹⁷⁰ (itself associated with both CVD and Alzheimer’s). Of note, 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 ^171,172, blood–brain barrier (BBB) dysfunction and Aβ transport to the brain ¹⁷³; if corroborated in humans this could underlie the epidemiologic association between SFA intake and Alzheimer’s ¹⁷⁴. Reciprocally, dementia and plasma Aβ₄₀ are associated with CVD ¹⁷⁵, while Aβ_40/42 binding to native or modified LDL enhanced foam cell formation in vitro ¹⁷⁶. In healthy adults high fat meals (vs. low fat) can also induce foamy-activated monocytes in association with postprandial TRLs ^177–179 and VLDL lipid saturation ¹⁸⁰; tested in vitro TRLs from meals high in SFAs (vs. MUFAs and n-3 PUFAs) induce greater immune cell apoB₄₈ (chylomicron) receptor expression, lipid accumulation and activation ^181–183, and coronary smooth muscle cell invasion ¹⁸⁴. Similarly, TRLs and their lipolysis products (mainly fatty acids) from LDLR^–/– mice on a diet rich in dairy fat/SFAs (vs. extra-virgin olive oil and nuts) induced more monocyte lipid accumulation ¹³⁰. 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 ⁴⁸. However, enrichment of chylomicron remnants with SFAs (vs. various UFAs) may lower hepatocyte LRP1 gene expression and uptake ¹⁶⁹, while inducing macrophage lipid accumulation ¹⁸⁵. Therefore the SFA content of TRLs may contribute to their atherogenicity.

More broadly, dietary fat quality may differentially affect systemic fat oxidation and storage. In human fatty acid tracer studies whole-body oxidation of (individual dietary) C16:0/18:0 SFAs is lower than C18-MUFAs/PUFAs, consistent with animal and cell studies ^186–188. Even after a 5-week stearate-rich diet postprandial stearate oxidation was still lower than oleate ¹⁸⁸; although stearic acid (vs. low fat and C16:0) was also shown to acutely induce mitochondrial fusion (in neutrophils) and lower plasma acylcarnitines (i.e. C18:0 and combined C16:0/18:0/18:1) ¹⁸⁹, suggesting it might increase oxidation of other fatty acids. In a more physiological context, short-term low carbohydrate/high fat diets favouring UFAs (vs. SFAs) also induce higher serum ketones ^105–107 (and improve long-term seizure control ¹⁰⁸), consistent with preclinical studies on hepatic β-oxidation and ketogenesis ^190,191. Further, in short-term imaging trials on normal ¹⁹² and overweight adults SFAs induce more liver fat (i.e. intrahepatic triglycerides) under isocaloric (i.e. butter/SFAs vs. sunflower oil/n-6 PUFAs ¹⁹³) or hypercaloric conditions (i.e. palm oil/SFAs vs. sunflower oil/n-6 PUFAs ^192,194; or various SFAs vs. UFAs and sugars ^195,196), while increasing the plasma SCD index (a putative marker of hepatic desaturation/DNL) ^192–194 and adipose lipolysis ¹⁹⁶, both of which may increase hepatic fatty acid availability. And inversely, in NAFLD, replacing ghee/SFAs with rapeseed oil/UFAs for 12 weeks reduced the steatosis grade, blood lipids and body weight ¹⁹⁷, while in a secondary analysis of an RCT with multiple diets (i.e. standard care, low carbohydrate or intermittent fasting) reductions in liver fat and stiffness correlated increased plasma n-6 PUFAs and decreased intake of SFAs/MUFAs, respectively ¹⁹⁸.

Cardiometabolic diseases typically involve insulin resistance and consequent hyperglycaemia, which is itself associated with CVD ¹⁹⁹; even in the nondiabetic PESA cohort HbA1c (i.e. monthly glucose control) independently correlated the presence and extent of subclinical atherosclerosis ³⁷. In T2D hypoglycaemic drugs have induced regression of coronary plaque ²⁰⁰, as well as carotid intima–media thickness (cIMT) in relation to postprandial glucose ²⁰¹. Accordingly, hyperglycaemia can induce oxidative-inflammatory activity and endothelial dysfunction ¹⁹⁹, while glycation of LDL increases arterial proteoglycan binding ^202,203. Insulin itself also normally suppresses VLDL-triglyceride secretion and promotes apoB catabolism and clearance ^165,204, and may modulate many atherogenic cells ^205,206. Interestingly, 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 ²⁰⁷. Indeed glucose-insulin homeostasis is affected by diet quality; in a systematic meta-analysis of over 100 controlled feeding trials isocaloric replacement of SFAs with PUFAs particularly improved insulin sensitivity and glucose control ¹⁹. Muscle biopsies from people spanning the range of insulin sensitivity (i.e. athletes–lean–obese–T2D) also report accumulation and subcellular localisation (i.e. sarcolemma and organelles) of saturated triglycerides ²⁰⁸ and sphingolipids/ceramides ²⁰⁹ correlates insulin resistance, while SFA intake was increased in T2D ²⁰⁸. Linking these observations, in some trials on healthy adults a higher palmitate/SFA intake (vs. oleate/MUFA) for 2–3 weeks ^210,211, or as a single bolus ²¹², induced blood/muscle sphingolipids/ceramides and suppressed glucose metabolism and insulin sensitivity. Also, in trials on overweight adults overfeeding SFAs from mixed sources (vs. UFAs or sugars) for 3 weeks ¹⁹⁶, or palm oil/SFAs (vs. sunflower oil/n-6 PUFAs) for 8 weeks ¹⁹⁴, increases multiple plasma/LDL sphingolipid species (opposite to PUFAs), paralleling induction of insulin resistance and liver fat. Further, 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 hypertriglyceridemia ²¹³.

Serum ceramides (esp. Cer16:0, Cer18:0 and Cer24:1) also predict CVD risk independent of conventional risk factors (incl. apoB), and are particularly elevated in obesity and T2D ²¹⁴. However, LDL ceramides were only elevated in T2D and can induce macrophage activation and muscle insulin resistance ²¹⁵, involving mitochondrial dysfunction ²¹⁶. Besides glucose metabolism, sphingolipids may have more direct effects on atherogenesis. Indeed arterial lesions were long known to contain sphingolipids ⁴¹; more recently sphingomyelin and several ceramides were increased in carotid plaques from symptomatic patients and diabetics, and induced smooth muscle cell inflammation and apoptosis in vitro ²¹⁷. Also, in endothelial cells endogenous ceramide can suppress nitric oxide (via AKT/eNOS) ²¹⁸ and increase the uptake and retention of oxLDL ²¹⁹, while LDL can deliver ceramide to mediate apoptosis ²²⁰. Moreover, aggregated LDL from human plaques was highly enriched in ceramide, which can result from sphingomyelin cleavage by SMase, promoting LDL aggregation and fusion in vitro ⁶². In people with established CAD, an assay measuring the susceptibility of LDL to aggregation (via incubation with human S-SMase) predicted CVD death independent of traditional markers (e.g. LDL-C), and such aggregated LDL activated macrophages and T cells in vitro (contrasting oxLDL) ¹³³. Aggregation susceptibility 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) ¹³³. In a subsequent trial on overweight adults, overfeeding by 1000kcals/day as SFAs from mixed sources for 3 weeks increased LDL sphingolipids and aggregation susceptibility, while UFAs (i.e. 57% MUFAs, 22% PUFAs) decreased LDL proteoglycan binding (and apoE) and sugars were without effect ¹³⁴. A further study with liver biopsies reported that LDL aggregation and lipid composition correlates the liver lipidome, implicating hepatic sphingolipid metabolism in LDL composition ²²¹. 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 ²²²). 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 ²²³.

Oxidation

Already in the 1950s lipid oxidation was detected in human plaque, and much subsequent research supports the involvement of lipid and protein oxidation in atherosclerosis ²²⁴. Native and oxLDL were even found in fetal aortas with and without macrophages, suggesting an early event ²²⁵. OxLDL can also be detected in plasma where it normally represents a very small fraction of LDL ²²⁶ and associates with CVD ^227,228, although not always independently of apoB (e.g. CHD ²²⁹ and MetS ²³⁰), likely due to 4E6 antibody cross-reactivity ²³¹. On the other hand, oxidised phospholipids on apoB₁₀₀ (oxPL–apoB) are independently associated with CVD and mainly carried by lipoprotein(a), an LDL variant; indeed oxLDL donates its oxPL to lipoprotein(a) in vitro ²³¹. Conversely, dietary and plasma antioxidant nutrients (esp. vitamins C, E and carotenoids—largely reflecting fruit/veg and seed oil intake) are inversely associated with CVD ^232,233, although the results of RCTs with high-dose (i.e. supra-physiological) supplements in general populations have mostly failed to show benefit ^224,234 (unlike general lipid-lowering). However, there is far less outcome data on more physiological antioxidant repletion/optimisation approaches and other phytochemicals (e.g. polyphenols) ²³⁴.

Interestingly, plasma oxLDL and oxPL–apoB increase transiently with statins in humans, and preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ^231,235. The high antioxidant capacity of plasma also suggests lipoprotein oxidation may occur elsewhere, such as within the arterial wall. Various catalysts are used in vitro, but most typically LDL is incubated with copper (Cu²⁺) sulfate, a transition metal mediating 1-electron oxidations ²²⁴. LDL oxidation induces many pro-atherogenic endothelial/inflammatory effects (e.g. eNOS ⁶⁶, CCL20 ²³⁶, EPCs ⁹⁷ and HSPCs ⁷²), and most characteristically, macrophage uptake and cholesterol loading via scavenger receptors ²²⁸. Further, unlike native LDL, macrophage uptake of oxLDL results in lipid trapping within lysosomes ²³⁷, cholesterol crystallisation and NLRP3 activation ^100,238. Similar oxidation of HDL also induces macrophage uptake, reversing its protective activity ²³⁹. In human tracer studies with autologous native and copper-oxidised LDL, the latter was cleared more quickly from plasma (T_1/2=85.8 vs. 124mins) but also detected more frequently (at 1hr) in areas of carotid lesions ²⁴⁰. In a subsequent study advanced carotid plaques (AHA type VI) were excised (at 24–72hrs) post-injection of labelled native LDL and revealed accumulation in foam cells specifically, which was suppressed in those on 4 weeks of high-dose α-tocopherol (aka. vitamin E—the most abundant lipophilic antioxidant) ²⁴¹.

However, despite much data apparently supporting a causal role of oxidative stress in atherogenesis, the general failure of high-dose vitamin E/antioxidant trials to improve hard outcomes suggests more complexity ²²⁴. Foremost, oxidation by free transition metals may have limited physiological analogy; although heme-iron (Fe²⁺) dysregulation may promote oxidation during advanced plaque haemorrhage and haemolysis ²⁴². On the other hand, human lesions express LOXs (non-heme iron-dependent dioxygenases), which oxidise PUFAs and can induce similar lipoprotein modification as copper oxidation ^243,244, but are not blocked by vitamin E ²⁴⁵. Human plaques also express iNOS ^246,247 and MPO ²⁴⁸ (both heme-dependent enzymes), while recovered LDL and apoA-I/HDL are highly enriched in their 2-electron protein oxidation products (i.e. nitrotyrosine and 3-chlorotyrosine, respectively) ^249,250, implicating immune-dependant redox modifications ²²⁴. These pathways are also not blocked by vitamin E ²⁵¹ (or serum ²⁵²), and resulting NO₂–LDL stimulates macrophage uptake and loading via scavenger receptor CD36 ^252,253, while MPO-modified tryptophan residues within apoA-I/HDL inactivate its ABCA1-dependent acceptor activity ^250,254. However, other data present yet further challenges ^90,224. For instance, in early studies LDL isolated from plaques was not always sufficiently oxidised for receptor-mediated uptake; rather LDL from human aortic fatty streaks and plaques exhibited increased macrophage uptake in a non-saturable manner attributable to aggregates ⁶⁸. Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^50,68 and CE accumulation beyond native or oxLDL ^255,256. Accordingly, the ability of copper-oxidised LDL to induce lipid droplets may be somewhat limited by defective lysosomal processing (prior to cholesterol esterification) ²³⁷. More ‘minimally’ oxidised LDL still exhibits atherogenic effects, such as a tendency to aggregate ⁶² and induce lysosomal crystals and NLRP3 activation ^100,238, so may contribute in these ways ⁶⁸. On the other hand, a systematic study of LDL oxidised with copper for 0.5–24hr showed that mild oxidation (>30min) initially inhibits macrophage selective CE uptake and native LDL-induced foam cell formation in relation to apoB fragmentation, before more extensive oxidation (>3hr) induces aggregation, CE oxidation and particle uptake ²⁵⁷. Furthermore, mild oxidation of HDL by copper, ALOX15 or HOCl (i.e. the product of MPO) actually increases efflux capacity by promoting formation of pre-β-migrating particles ²⁵⁸. Also, human ALOX15 variants if anything suggest increased enzyme activity is athero-protective ²⁵⁹, consistent with ALOX15 overexpression increasing reverse cholesterol transport ²⁶⁰.

Considering the specificity and spatiotemporal pattern of lipid oxidation in plaque may provide some reconciliation. Of lipids both UFAs and cholesterol are susceptible to oxidation at their double bonds, of which PUFAs have many and linoleic acid (C18:2n-6) is most abundant in plasma and plaque CEs ⁴¹. Accordingly, comprehensive analysis of CEs from human peripheral vascular plaques revealed a substantial proportion are oxidised (avg. 21%), with cholesteryl linoleate to the greatest extent (i.e. C18:2 > C20:4 > C22:6), and the most abundant species being mono- and di-oxygenated derivatives of linoleate (i.e. HODEs and HpODEs, respectively) ²⁶¹. The HODE-CE profile exhibited no regio- or stereo-specificity suggesting a dominance of non-enzymatic oxidation (vs. 15-LOXs), although triglyceride PUFAs were not oxidised indicating some specificity ²⁶¹. In several earlier studies 13-HODE stereoisomer ratios were consistent with ALOX15 activity, particularly in early lesions ²⁶², while more recent studies on carotid plaques found increased expression of ALOX15B only, and in association with macrophages and HIF-1α ²⁴⁴. Recent high-resolution imaging of advanced carotid plaques also found oxidised CEs co-localise with sphingomyelin in the necrotic core, while a metabolite resembling 7-ketocholesterol (representing 1-electron cholesterol oxidation) was uncorrelated ²⁶³. Several earlier studies also found plaque lipid oxidation occurred despite normal levels of α-tocopherol ²⁵¹, and was similar in T2D ²⁶⁴. In particular, an analysis of intimal lipoprotein-containing fractions of human aortic lesions from early to late-stage disease found accumulation of cholesterol (AHA types II–III) and CEs (types IV–V) preceded their major oxidised derivatives (i.e. 27-hydroxycholesterol and CE hydro(pero)xides, respectively), while 7-ketocholesterol only increased at late stages (types V–VI), and α-tocopherol and CoQ₁₀ levels remained relatively stable throughout ²⁶⁵. Another stage-dependent analysis of whole aortic lesions included tocopherol oxidation products and implicated 2-electron (enzymatic) oxidants ²⁶⁶.

These observations contrast typical conditions in vitro ²²⁴, where under strong copper-oxidising conditions α-tocopherol acts as a chain-breaking antioxidant and its depletion underlies the lipid oxidation lag phase and formation of secondary/advanced oxidation products (e.g. aldehydes, isoprostanes, etc.) ²⁶⁵. Conversely, under more mild conditions the α-tocopherol radical can initiate lipid oxidation ²⁶⁷, especially when there are insufficient regenerative co-antioxidants (e.g. CoQ₁₀ and carotenoids) ²⁶⁸. Further, copper-oxidation of LDL generates substantial 7-ketocholesterol, which in macrophages inhibits lysosomal SMase causing accumulation of sphingomyelin–cholesterol particles ²³⁷, and dose-dependently induces cholesterol crystals ²⁶⁹, thereby mediating key effects of oxLDL. However, in vivo 7-ketocholesterol is quantitatively and temporally overshadowed by 27-hydroxycholesterol, which is produced by sterol 27-hydroxylase, indicating a dominance of enzymatic oxidation ²⁶⁵. In the liver this pathway initiates bile acid synthesis, while elsewhere it may increase the polarity of cholesterol and facilitate efflux, especially when HDL is deficient, before return to the liver for further conversion and excretion ²⁷⁰. In human lesion macrophages increased expression of sterol 27-hydroxylase is accompanied by the expression of genes functionally linked in vitro; specifically, RXR and PPARg ligands induce sterol 27-hydroxylase ²⁷¹, which in turn induces 27-hydroxycholesterol and LXR—an oxysterol sensor mediating efflux ²⁷². Further, the early linoleate oxidation metabolite 13-HODE is a natural PPAR ligand ²⁶² and also induces macrophage efflux to apoA-I via a PPARα/g–LXRα pathway ²⁷³. Upstream, 15-LOXs can directly oxidise CE-PUFAs, which are also preferred substrates for hydrolysis, and reincorporated into phospholipids ²⁴⁴; while ALOX15 specifically is induced by Th2/M2 cytokines and apoptotic cells (via LXR ²⁷⁴), consistent with a role in lipid/tissue homeostasis ^243,244. Therefore, considering all the above, mild enzymatic oxidation of trapped lipids may support cholesterol clearance ^262,265,275, whereas excessive oxidation may favour lipid trapping via 7-ketocholesterol ²³⁷ and inactivation of apoA-I/HDL ^250,254,258, perhaps in relation to advanced disease and inflammation ²²⁴.

The effect of dietary fats on lipoprotein oxidation has been tested since the early 90s in short-term trials; here MUFA-rich diets (vs. n-6/n-3 PUFAs) can typically lower LDL and HDL oxidation, and susceptibility to copper oxidation (i.e. lag time and/or rate) and monocyte adhesion in vitro, which correlates plasma oleate/linoleate ratios ^276–278. This has fuelled some concern, although doesn’t mirror hard outcome data or reflect the more nuanced redox biology in vivo, as above. Accordingly, in men ¹¹⁰, monkeys ¹²² and mice ¹²⁴ n-6 PUFA-rich diets increase linoleate/oleate ratios in plasma and plaques, and oxidation in vitro ¹²² and in vivo ¹²⁴, yet are protective. In humans dietary linoleic acid (vs. SFAs) increases plasma primary oxidation metabolites (i.e. HODEs and oxoODEs) ²⁷⁹, but also potentially efflux via LXRα/ABCG1 expression ^26,114. 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 ^280,281. Moreover, the food matrix is also important. For instance, a 3-week diet of 31% sunflower oil/n-6 PUFAs (vs. olive oil/MUFAs) lowered LDL levels, oxidation susceptibility and proteoglycan binding, in relation to LDL antioxidant content and size ²⁸². Further, in healthy adults an n-6/n-3 PUFA-rich walnut meal (i.e. 59g fat, 42g PUFAs) increased postprandial antioxidant capacity and lowered malondialdehyde (5-hour AUC) and oxLDL (at 2 hours) ²⁸³, while longer trials show enrichment of PUFAs with preservation of oxidation status ^284,285, alongside many other cardio-protective effects (reviewed in ²⁸⁶). Conversely, food storage and processing can oxidise lipids prior to ingestion. When fed to humans oxidised linoleic acid could be detected in chylomicrons/remnants for 8 hours, whereas oxidised cholesterol appeared in all major lipoproteins and persisted for 72 hours; tested in vitro oxidised cholesterol was transferred to LDL and HDL, potentially via CETP ²⁸⁷. In animal models both dietary oxidised linoleic acid and cholesterol promoted atherosclerosis ²⁸⁷; although oxidised linoleic acid has also been reported to lower blood lipids and atherosclerosis, presumably in relation to more mild oxidation and 13-HODE ²⁷³. Of note, in a unique RCT comparing high quality to oxidised fish oil (approximating some commercial supplements), only the former lowered apoB-lipoproteins ²⁸⁸. The gut is another potentially relevant site of redox activity ²⁸⁹. For instance, in humans and mice red meat ingestion induced postprandial plasma lipid oxidation and corresponding LDL–malondialdehyde modification, which was greatly inhibited by polyphenols ^290,291; in gastric models this was also inhibited by olive oil/MUFAs, opposite to fish oil/n-3 PUFAs ²⁹². Indeed the stomach has been conceptualised as a bioreactor, which denatures foods and facilitates redox chemistry, and where heme-iron can deplete antioxidant vitamins and induce advanced lipid oxidation ²⁸⁹, as with copper-oxidation in vitro.

Despite their apparent oxidative stability, SFA-rich diets (vs. carbohydrates or MUFAs) may also increase LDL susceptibility to oxidation in vitro in relation to LDL MUFA/PUFA ratios ^277,278 and APOE promoter variants ¹²¹. Further, excess dairy fat may favour oxidation 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 flow-mediated dilation (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 ¹⁷⁷. 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 ²⁹³; and in another study replacement with olive oil and nuts lowered monocyte oxLDL uptake and CD36 expression, which was modulated correspondingly by TRLs from each diet ¹³⁰.

Lipopolysaccharides

In 1999 the Bruneck (prospective) 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 ²⁹⁴. Nowadays many studies support a link with cardiometabolic disease and athero-thrombosis (e.g. reviews ^295–297). LPS is the canonical ligand for toll-like receptor 4 (TLR4), which stimulates innate immunity and primes the NLRP3 pathway ^43,100,238; although depending on source/structure, it can act as an agonist or antagonist (e.g. E. coli and Bacteroides, respectively) ²⁹⁸. In mouse models of endotoxemia platelet TLR4 triggers neutrophil extracellular traps (NETs) to ensnare bacteria in liver sinusoids and pulmonary capillaries ^299,300; although in APOE^–/– mice LPS-induced neutrophils also promote monocyte recruitment and aortic atherosclerosis ^301,302, and increase carotid plaque MPO and instability ³⁰³, consistent with human samples ^302,303. 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 ³⁰⁴. Further, carotid LPS correlated plasma LPS (r=0.668), which correlated soluble TLR4 and serum zonulin (a marker of intestinal permeability) ³⁰⁴; with similar blood marker relationships reported in other populations ^305–307. 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) ^308–310. Butyrate is the archetypal beneficial short-chain fatty acid (SCFA) and ameliorates atherosclerosis in APOE^–/– mice by lowering gut permeability and endotoxemia ³¹¹ and inducing ABCA1-dependent cholesterol efflux ³¹². Several human studies also find depletion of Bacteroides spp. ^308,313,314, which when administered to APOE^–/– mice also lowered atherosclerosis and gut/blood LPS ³¹³.

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 ³¹⁵, transferred from HDL to LDL (via LBP and PLTP) ^316,317, and removed predominantly via the hepatic LDLR (in humans) ^318,319. A single LDL particle can bind many LPS molecules with only minor changes to its composition ³²⁰; such binding sequesters the lipid A region within the phospholipid monolayer and hepatic uptake is apparently non-toxic ^318,319. 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 ³²¹, contrasting the situation in LDLR^–/– mice ^321,322. Importantly, lipoproteins may also carry bioactive LPS into other tissues to elicit inflammation (e.g. endothelium ^323,324, adipose ^325,326 and brain ^327,328), 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 ³²⁹. Here LPS can activate endothelium and monocyte chemotaxis ^314,323,324, induce smooth muscle synthesis of elongated proteoglycans (equal to traditional agonists) ³³⁰, increase LDL susceptibility to oxidation (by copper, endothelial and smooth muscle cells) ³³¹, and stimulate macrophage oxLDL uptake and foam cell formation ³³². Moreover, in humans and rodents LPS impairs total (i.e. macrophage–faeces) reverse cholesterol transport at multiple steps ³³³; in part via induction of MPO/SAA ³³⁴ and suppression of ABCA1 ³³⁵.

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) ³³⁶. In systematic reviews dietary fat quality also affects gut microbiota ²³ and plasma LPS ²². Of interest herein, habitual SFA intake has been associated with bile-resistant sulfide-producing genera such as Bilophila (family Desulfovibrionaceae) in faeces ³³⁷ and colonic mucosa ³³⁸. Also, in a 3-week trial on overweight adults (similar to those above ^134,196), overfeeding SFAs increased faecal Gram-negative Proteobacteria (mainly via Desulfovibrionaceae), while UFAs increased butyrate-producing bacteria (i.e. Lachnospira, Roseburia and Ruminococcaceae spp.) ^195,196; similarly, replacement of butter/SFAs with margarine/n-6 PUFAs for just 3 days induced Bifidobacteria and Lachnospiraceae ¹⁶³. In mice dairy fat/SFAs (vs. safflower oil/n-6 PUFAs) induce secretion of taurine-conjugated bile acids to support expansion of Bilophila wadsworthia (a sulfite-respiring bacterium) ³³⁹, which in turn lowers colonic butyrate and induces LPS biosynthesis and translocation ^339–341. 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 ²². 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 ³⁴²; although an intermediate effect was significant in a prior pig study ³⁴³. 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 ^344–346; an effect quicker in obesity ³⁴⁶ and still present after 12 weeks of a SFA-rich diet ³⁴⁵. 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 ³⁴⁷. Accordingly, in preclinical studies LPS absorption can occur via chylomicrons ³⁴⁸, which deliver bioactive LPS to lymph ³⁴⁹. 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 ^350–352. Whereas n-6 PUFAs (vs. SFAs) induced PBMC TLR4 expression in humans ¹¹⁴ and increased macrophage LPS sensitivity in mice ¹²⁵, although this may be moderated by long chain n-3 PUFAs ³⁵³, implicating n-6/n-3 balance. Nevertheless, 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 ³⁵⁴ and implicated in pathogenesis (e.g. reviewed in ³⁵⁵). As above, SFA-rich diets can induce liver fat; in particular, one group found this occurred with increased adipose lipolysis and inflammation (i.e. tissue transcriptome), blood liver enzymes, ceramides and endotoxemia (i.e. LBP/CD14 ratio), faecal Proteobacteria ¹⁹⁶ and baseline abundance of Bilophila ¹⁹⁵. Similarly, in mice an obesogenic diet rich in palm oil/SFAs (vs. sunflower oil/MUFAs) induced adipose macrophages and hepatic inflammation, and while also enriching small HDL in acute-phase proteins (incl. SAA) and lowering liver­–faeces cholesterol transport ¹⁶², akin to low-dose LPS ³³⁵. Further, Bilophila wadsworthia aggravated dairy fat/SFA-induced metabolic dysfunctions and steatosis, while suppressing microbial butyrate and inducing LPS ³⁴⁰. In the postprandial setting, in young healthy adults a single coconut oil/SFA-rich meal suppressed HDL anti-inflammatory activity (on TNFα-activated endothelial cells) and FMD, while the former improved after safflower oil/n-6 PUFAs ³⁵⁶. A single bolus of palm oil/SFAs (equivalent to a SFA-rich meal) also 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 with hepatic transcriptome analysis revealed evidence of LPS/TLR signalling ³⁵⁷. Of interest here, fat-induced LPS absorption may not only involve chylomicrons, but a more rapid and dominant portal vein pathway ³⁵⁸, wherein intestinal secretion of HDL₃ binds LPS and restrains high fat/lard-induced liver injury and fat storage ³⁵⁹. For further context, in other postprandial trials cream-induced liver fat was attenuated by co-administration of 50g glucose but not fructose ³⁶⁰, which itself may also be capable of inducing endotoxemia ³⁶¹, DNL and ceramides ³⁶². This unique effect of glucose may involve its ability to stimulate insulin and thereby inhibit adipose lipolysis and fatty acid flux to the liver ³⁶⁰. 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 ³⁶³. Such endotoxemia also induces ceramides in VLDL and LDL ³⁶⁴, which in rodents is accompanied by activation of S-SMase in serum and de novo sphingolipid biosynthesis (i.e. SPT) in liver ^364,365. Therefore LPS may mediate some of the differential immuno-metabolic effects of SFAs herein.

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 ³⁶⁶. Such rafts may serve as functional platforms to assemble proteins subserving cell signalling and endocytosis ³⁶⁷, which can be modulated by exogenous lipids. In particular, free cholesterol in the lipoprotein monolayer is in equilibrium exchange with cell membranes ³⁶⁸, 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) ³⁶⁹. Further, the hepatic LDLR is associated with both clathrin and caveolae-rich membrane regions ³⁷⁰ (which correspond to non-raft and raft regions, respectively ³⁶⁷), and treatment with LDL or cholesterol induced translocation to caveolae coinciding with reduced LDL uptake ³⁷⁰. In non-hepatic cells ⁴⁸ internalised LDL-C also travels from lysosomes to plasma membrane first before ER regulatory domains ³⁷¹. And in the other direction, the ABCA1 transporter may associate with cholesterol-rich lipid rafts ³⁷² to mediate efflux to apoA-I/HDL ³⁷³; indeed the composition of nascent HDL resembles lipid rafts ³⁷³. 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 ³⁷⁴. 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 ³⁷⁵. In monkeys corn oil/n-6 PUFAs (vs. coconut oil/SFAs) increased LDL uptake by PBMCs which correlated membrane fluidity and lower plasma cholesterol ³⁷⁶; 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 ³⁷⁷. Fatty acid fluidity might also affect lipoprotein packing and surface protein conformation ¹⁵¹, as well as lipid droplet hydrolysis and cholesterol efflux ^122,276,378.

Lipid microdomains are directly implicated in vascular function and atherogenesis. Firstly, LDL induces transcytosis of macromolecules through endothelium via the LDLR, cholesterol and caveolae ⁹⁵. In human atherosclerotic plaques the oxLDL receptor LOX-1 is also associated with caveolae and dissociated by statins or MβCD (which extracts membrane cholesterol), thereby abrogating oxLDL-induced apoptosis ³⁷⁹. As above, LDL can also deliver ceramide to endothelial cells ²²⁰, where endogenous ceramide promotes the uptake and retention of oxLDL via regulation of transcytosis-related and raft-associated proteins, including LOX-1 ²¹⁹. Further, hypercholesterolemia and LDL inhibit endothelial nitric oxide synthase (eNOS) via translocation to caveolae rafts (reviewed in ⁶⁶). Hypercholesterolemia and 7-ketocholesterol also induce endothelial A-SMase/ceramide-dependent membrane raft redox signalling platforms linked to NLRP3 activation ³⁸⁰. Notably, electronegative LDL also possesses intrinsic SMase activity associated with apoB₁₀₀ serine O-glycosylation ³⁸¹; this may be outward-facing so as to engage plasma membrane sphingomyelin, generating ceramide-based microdomains and endocytic vesicles ^382,383. Moreover, arterial SMase can hydrolyse sphingomyelin within lipoproteins themselves generating ceramide-rich domains, which may act as nonpolar spots promoting aggregation via hydrophobic interaction ⁶², as well as displacement and release of cholesterol to neighbouring vesicles ³⁸⁴. Atherosclerotic plaques were also reported to contain membranes enriched in free cholesterol and crystalline domains ⁶⁶. In preclinical studies plaque crystals co-associated with cholesterol microdomains ³⁸⁵, which can be shed from macrophage membranes ³⁸⁶. Rapid loading of macrophages via phagocytosis of large lipid droplets induces lysosomal free cholesterol and extracellular crystals ⁵⁶; 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 ^387,388. Further, lipid oxidation induces crystalline domains in model membranes under conditions of hyperglycaemia, which can be inhibited by n-3 PUFAs (esp. EPA) ³⁸⁹. 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 ³⁹⁰.

Regarding immuno-pathogenesis, in human cohorts LDL-C correlated a haematopoietic monocyte skewing (vs. granulocytes) in blood ³⁹¹ and proinflammatory macrophage phenotype in adipose ³⁹², 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 ³⁹³. 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) ³⁴⁶. Of relevance here, in LPS-stimulated macrophages TLR4 activation requires cholesterol biosynthesis (via FASN) to enter lipid rafts ³⁹⁴, while ABCA1-dependent cholesterol efflux suppresses raft-associated TLR/inflammatory signalling in macrophages ^395,396 and endothelial cells ³⁹⁷.

Shortly after the discovery of TLR4 as the LPS receptor it became apparent that fatty acids could also modulate TLR signalling ³⁹⁸. 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 ^399,400, and potentiate that by TLR ligands, all of which is inhibited by UFAs (esp. DHA) ^398,401. 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 ⁴⁰², while hypo-acylation or incorporation of UFAs result in antagonist activity ^398,401; similarly, total gut LPS silences TLR signalling due to hypo-acylated lipid A in Bacteroidales ²⁹⁸. Cellular sensitivity to low levels of LPS is supported by initial binding to surface CD14/CD36, which facilitates transfer to the TLR4–MD2 complex ⁴⁰³, wherein its saturated acyl chains interact with MD2 lipid domains inducing TLR4 dimerization ⁴⁰¹. As a corollary, free fatty acids (SFAs and UFAs) may also bind within the hydrophobic pocket of MD2 to directly modulate TLR4 signalling ^401,404,405, while other evidence suggests palmitate acts indirectly via lipid metabolism and ER stress ⁴⁰⁶. 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 ³⁵⁰. In line with this, in hyperlipidemic mice systemic inhibition of the ER-associated enzyme SCD1, which mediates endogenous desaturation of SFAs to MUFAs, induced macrophage TLR4 hypersensitivity ⁴⁰⁷. Since plasma membrane rafts are rich in polar lipids with saturated acyl chains and can be modulated by SCD1 ⁴⁰⁷ and n-3 PUFAs ⁴⁰¹, this may contribute to general effects. LPS and oxidative stress-induced raft–TLR4 complex formation also requires A-SMase-derived ceramide ^408,409 (which has structural similarities to lipid A ⁴¹⁰), while palmitate augments LPS inflammatory responses via SMase and de novo ceramide synthesis ^411–414. Free SFAs (i.e. palmitic and stearic acid) can also activate macrophage NLRP3 inflammasomes via flux into phosphatidylcholine and ER stress ⁴¹⁵, and even crystallisation ⁴¹⁶, 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 ⁴¹⁷.

Lipid rafts are also involved in endocytosis ³⁶⁷ and represent a common entry point for many viral, bacterial and fungal pathogens ^418–420. For instance, in the colon butyrate may inhibit enteric pathogen invasion via depletion of cholesterol and increased membrane fluidity ⁴²¹. Similarly, in porcine ileum samples SFA-induced LPS permeability was abrogated by MβCD, implicating lipid rafts ³⁴³. Another study using oleate and taurocholate (which is especially induced by SFAs ³³⁹) further implicated a raft/CD36-dependent pathway ³⁵⁸. 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 ⁴²², and induction of faecal/plasma LPS by a high fat/lard diet ⁴²³. The differential effects of fatty acids on TLR4 signalling ³⁹⁸ also parallels those on postprandial LPS ³⁴³.

Ecology

Evidence of atherosclerosis has been reported in ancient humans spanning 4000 years and from diverse locations suggesting a basic predisposition ⁴²⁴; 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 ³⁸. These populations may have been exposed to various enduring risk factors, including diet, smoke and infections, although this remains speculative ⁴²⁵. However, there are some notable examples of extant pre-industrial people with divergent health outcomes, suggesting post-industrial changes are also important ⁴²⁶. 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 ⁴²⁷; as well as atrial fibrillation ⁴²⁸, age-related brain atrophy ⁴²⁹ and dementia ⁴³⁰.

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/or diet ^88,89,122; although important metabolic differences remain (e.g. LDLR/APOE knockout ⁸⁹, no CETP ⁸⁸, ACAT2 expression ^46,136, LCAT specificity ^143,144, LOX specificity ²⁴⁴ and CYP27 regulation ²⁷²). Further, unlike small animals, in human arteries plaque-prone regions exhibit diffuse intimal thickening, which is initiated in utero ^44,61. Presumably these adaptations augment vessel strength and elasticity in response to mechanical stress, while also seeding the soil for atherogenesis ^44,54,61. Already in the human foetus arterial lesions occur in relation to maternal hypercholesterolemia and intimal LDL accumulation ⁴³¹. Under physiological conditions endothelial transcytosis of lipoproteins may support vascular lipid metabolism and immunity ^52,53, but since cholesterol is not catabolised here this creates a vulnerability to accumulation under hypercholesterolemia. In particular, endothelial ⁹⁸ and smooth muscle cells ⁴³² treated with native or aggregated LDL, respectively, seem to have a limited capacity for efflux compared to myeloid cells, and during atherogenesis may be an early site of crystallisation, driving inflammation ^67,98. Moreover, experimental atherosclerosis requires NLRP3 ¹⁰⁰, suggesting a lipid–immune vicious cycle and implicating other cell stressors. Foremost, many infections are associated with atherosclerosis and the acute-phase response induces metabolic changes implicated in CVD, which may support immunometabolism, signalling and defence ^433,434. For instance, infections/inflammation modulate systemic insulin sensitivity (i.e. glucose metabolism) ^435,436, lipid metabolism (e.g. lipolysis, cholesterol and sphingolipids) and lipoprotein modifications (e.g. oxLDL) ⁴³⁴. More specifically, macrophages switch to a glycolytic metabolism and accumulate lipid droplets with antimicrobial activity ⁴³⁷, which depend on CD36 (i.e. lipid import) ⁴³⁸. Further, LPS–TLR4 signalling requires cholesterol synthesis ³⁹⁴ and inhibits systemic reverse cholesterol transport ³³³, while various inflammatory mediators induce LDL transcytosis through endothelium ¹⁰¹, which may further increase lipid availability. Bacterial sequestration may be supported by vascular retention (e.g. LPS­–TLR4 ^299,330 and M. tuberculosis ⁵⁵) and phagocyte clearance by LDL binding (e.g. LPS ^331,332 and group A Streptococcus ⁴³⁹). These mechanisms could therefore support acute survival, while persistent stimulation exacerbates vascular disease hastening late-life mortality ⁴³⁴, as a form of antagonistic pleiotropy. Indeed while CVD is currently the leading cause of death, ancestrally it was likely infections and injury ⁴²⁶, 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 ⁴⁴⁰. The specific composition of LPS varies between bacteria and is modulated by environmental factors ⁴⁴⁰. In pathogenic bacteria the lipid A region is typically hexa-acylated with SFAs ⁴⁰², 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 ⁴⁴¹. 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 ⁴⁴². In mice SFA-rich diets (i.e. C12/16:0) exacerbated central autoimmunity by increasing Th1/17 activity via the small intestine ^443,444. Th17 cells express particularly high levels of TLR4, and LPS directly induces Th17 differentiation in vitro ⁴⁴⁵. 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 ⁴⁴⁶. 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 ⁴⁴⁷. This was consistent with SFA induction of innate immune memory (i.e. ‘trained immunity’), itself another double-edged sword ⁴⁴⁸. 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 ³⁹¹. Dietary cholesterol may also affect the pathophysiology of infectious and autoimmune disease (reviewed in ⁴⁴⁹).

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; indeed excess long-chain SFAs can induce cell stress/inflammasome pathways, which are offset by UFAs ⁴¹⁶ via increased membrane unsaturation ^375,415 and SFA channelling into triglycerides and β-oxidation ^450,451. Similarly, the ER-associated enzyme SCD mediates endogenous desaturation and protects from SFA toxicity in various cell types; and in human adipocytes DNL and SCD were functionally coupled, which may underlie the lipogenic effect of SFAs ⁴⁵². Accordingly, postprandial TRL fatty acids may induce foamy monocytes via ER-derived lipid droplets with increased unsaturation to protect from SFA toxicity ¹⁸⁰. This lipogenic cost is also illustrated in hyperlipidemic mice on SFA or MUFA-rich diets where deficiency of SCD suppressed obesity-related metabolic disorders and triglycerides, while inducing atherosclerosis, plasma SFAs and macrophage TLR4 hypersensitivity ⁴⁰⁷. Furthermore, despite SCD, the dietary SFA/MUFA ratio still modulates tissue ratios (and physiology) ^210,350 and we are especially limited at de novo PUFA synthesis, underlying the essential fatty acids (i.e. C18:2/3). Similar issues pertain to cholesterol, which is also fundamental to membrane physiology, 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 ^270,453) 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 ^135,141 and LCAT ^145–147), suggesting their availability may influence cholesterol turnover and therein processes of uptake/efflux, transport and crystallisation; interspecies differences in LCAT specificity for PUFAs may also correlate susceptibility to (diet-induced) atherosclerosis ¹⁴⁴. Differential modulation of gene expression may affect systemic metabolism and sterol excretion ^114,140, while in the gut modulation of microbial cholesterol ¹⁶³ and bile acid metabolism ³³⁹ may also be important. Further, the role of lipids and lipoproteins in immunity may superimpose another layer of regulation ⁴³⁴.

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 (e.g. total fat ~14–17% kcals; PUFA/SFA ratio ~0.9–1.7) ^454,455, after which our diet became increasingly diverse and animal-based with our spread to colder environments and pastoralism ⁴⁵⁶. Initially the hepatic LDL shunt pathway may have evolved to favour cholesterol conservation ⁴⁸, 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 PUFA/SFA ratio ^{111,112,116,135,139}. In parallel, the susceptibility of meat to deterioration might have also increased exposure to pathogens, consistent with our relatively low stomach acid pH (i.e. similar to scavengers) ⁴⁵⁶ and animal food aversion during early pregnancy (i.e. morning sickness), a relatively immune-suppressed period ^457,458. The advent of cooking would support sterilisation and may be reinforced by the appealing sensory qualities of advanced glycation end products (AGEs) ⁴⁵⁹, albeit at the potential cost of cardiometabolic dysregulation, as seen in modern humans ⁴⁶⁰. However, despite these physiological challenges native populations often exhibit relative cardiometabolic health ^426,461. 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 ⁴²⁶, with a low fat intake (i.e. median ~18% kcals) from plants and lean meats ⁴⁵⁶. Similarly, Tsimane vascular health is accompanied by a low LDL-C (esp. till 2011) ⁴²⁷ and fat/SFA intake (i.e. men: 15.1/3.7% kcals, respectively) from a plant-dominant diet with moderate fish/meat ⁴⁶². Furthermore, in this energy-limited and pathogenically diverse context, the ancestral APOE4 allele is actually associated with better cognition in those infected with parasites ⁴⁶³, 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 ⁴⁶⁴, suggesting it may not have the same deleterious effects as in post-industrial populations, but instead support cognition and immunity.

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 ⁴⁶² and plasma lipids ⁴²⁷ (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 ⁴⁶⁵ and an ‘affluent’ diet ⁴⁶⁶. In industrialised societies major SFA sources are now grain-fed meats (i.e. more fat and less n-3 PUFAs vs. grass-fed ⁴⁶⁷) and concentrated/added fats from animals and tropical plants, in the context of a diet high in ultra-processed foods/calories and low in micro-/phytonutrients, implicating evolutionary mismatch in SFA-associated diseases ⁴⁶¹. In trials herein unfavourable effects particularly occur with dairy fat, palm oil and coconut oil, all rich in cholesterol-raising and inflammatory C12–16:0 SFAs, although many nutritional and physiological factors may moderate this (and serve as study confounders). For instance, SFA-induced elevations in plasma cholesterol may depend upon intake of dietary cholesterol ^111,112, plant-based PUFAs ^135,139 and associated phytosterols ^160,161. SFA-induced postprandial inflammation may especially occur in obesity ^346,347,468, but be blunted by lipid-lowering therapy ³⁴⁶ or co-ingestion of phytochemicals (e.g. polyphenols ⁴⁶⁹, spices ⁴⁷⁰ and fibre ⁴⁷¹), 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 and exhibit robust anti-LPS/TLR effects ^{339,342,343,398,401} (not without potential for excess ^472,473), implicating omega-3 status. SFA-induced liver fat may be promoted by poor metabolic health ⁴⁷⁴, overfeeding ^{192,194–196} and excess fructose ³⁶⁰. Conversely, low carbohydrate diets may mitigate the differential effects of SFAs (vs. UFAs) on insulin sensitivity and inflammation, but not cholesterol, SCD and ketones ^105–109. Accordingly, low carbohydrate diets increase muscle fat oxidation ¹⁰⁵, which can protect muscle cells from palmitate toxicity in vitro ⁴⁵⁰; ketosis also has inherent anti-inflammatory effects ⁴⁷⁵ and a 3-day isocaloric ketogenic diet suppressed LPS/palmitate-induced NLRP3 inflammasome activation in macrophages in vitro ⁴⁷⁶.

Also noteworthy, over the past century consumption of seed oils/n-6 PUFAs has greatly increased and beyond what may be possible in pre-industrial diets ³⁰, but with a corresponding enrichment of blood/adipose associated with favourable CVD and cancer outcomes ⁴⁷⁷, seemingly creating an ecological disconnect. In the contemporary context however, such benefits are realised when dietary n-6 PUFAs/linoleate replace “carbohydrates” or SFAs/dairy fat ^3,5,13, which are also typically refined/concentrated from wholefood and evidently more maladaptive. In this regard, seeds feature in native ⁴⁵⁶ and ancient diets ⁴⁷⁸, with some tree nuts also being rich in n-6/n-3 PUFAs (e.g. walnuts, mongongo and pine nuts); and other primates consume a high proportion of PUFAs from fruit (>n-6) and leaves (>n-3) ^454,455. Nonetheless several trials herein of n-6 PUFAs (vs. SFAs) in humans ¹¹⁴ and mice ^123–125 do show some tissue-specific signs of inflammation. Of potential relevance, recent human studies suggest dietary linoleate may have opposite effects on serum CRP and adipose inflammatory gene expression in relation to FADS1 genotype ⁴⁷⁹. Linoleate intake can also affect peripheral long-chain n-3 status ⁴⁸⁰ and supplemental EPA bioavailability (vs. SFAs) ⁴⁸¹, presumably via competition for biosynthetic and esterifying enzymes; although in reciprocal long-chain n-3 intake can lower long-chain n-6 ⁴⁸², suggesting the importance of balance. Isolation of PUFAs from their natural food matrices may also facilitate oxidation during food processing and digestion, which could eventually abrogate benefits and provoke inflammation. From a wholefood perspective, plants (esp. nuts/seeds) can be rich in C18-MUFAs/PUFAs (n-6 and n-3, as above) and marine life in long-chain n-3 PUFAs, while terrestrial animals are lean, broadly consistent with current health associations in post-industrial people.

Overall, while many factors may promote atherogenesis, as a condition of arterial lipid accumulation, a lipid-threshold may ultimately govern its progression ^483,484. Accordingly, atherosclerosis infrequently occurs in mammals and humans with an LDL-C <80mg/dl ^55,368; including the Tsimane (with chronic inflammation) ⁴²⁷ and the middle-aged PESA cohort (without conventional risk factors) ³⁷. Moreover, decades of research on experimental atherosclerosis shows that most aspects of advanced plaques can regress, including necrotic and crystalline material, in association with dramatic lipid lowering, improved HDL function and M2 macrophage polarisation ^484–486. In human trials athero-regression can be induced with intensive lifestyle changes ⁴⁸⁷ (e.g. with <SFAs ^103,207) and/or lipid-lowering drugs ⁴⁸⁸ (i.e. with LDL-C <80mg/dl ^200,489), although is typically modest. However, some extreme cases have also been reported with CCTA ^490–493, which may be facilitated by younger plaque, less risk factors and lower lipids ⁴⁹⁴. In fact when considering other mammals, newborn humans and native populations, this physiology seems more normal ^368,495, 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 chronic deviation from physiological homeostasis as a result of unfavourable gene–environment interactions. In this regard, dietary fat quality may be important via effects on plasma lipids and the immuno-metabolic milieu (Box).

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