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. Further, 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 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 carbs (n=5, RR=0.84, 95% CI 0.67–1.06), but not mortality (albeit with 75% less death vs. event data) ¹⁴. Further supporting this, a diverse literature of shorter RCTs show replacement of SFAs can favourably modulate various biomarkers of risk and pathophysiology; e.g. lipids/lipoproteins ^15,16, immune-inflammation ^17,18, glucose-insulin homeostasis ¹⁹, liver fat ²⁰, endothelial integrity and platelet activity ^21,22.

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 ^23–26. As well as issues with food/nutrient replacements above, other nuances include the specific SFAs/PUFAs, food source/matrix, dietary patterns and endogenous biosynthesis (i.e. de novo lipogenesis, DNL), all of which may confer differential effects. These factors in mind, this post explores some of the major lipid-based pathways which may mediate the association of dietary SFAs with CVD, as guided by qualitative comparisons with UFAs in humans, and supported by preclinical and mechanistic models.

Cholesterol

CVD events are typically precipitated by atherosclerosis—the build-up of arterial plaque. 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 ²⁷, Scotland ^28,29, Spain ^30,31 and Egypt ³²), affecting many arteries (esp. aorta–iliac) and correlating brain hypometabolism ³⁰. 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) ³³, along with increasing leukocytes (esp. macrophages), necrosis, fibrosis and calcification ³⁴. 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 ^33,35–37.

Low-density lipoprotein cholesterol (LDL-c) in particular has a long association with CVD ³⁸; even in explicitly low risk populations LDL-c has been independently and linearly associated with subclinical atherosclerosis and CVD mortality ^31,39,40. 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) ^38,41. However, CVD risk generally tracks better with apoB, which reflects the particle count of all atherogenic lipoproteins—LDL being most abundant (typically >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) ^42,43. A recent Mendelian study further suggests the risk from apoB is actually mediated by non-HDL-c, which reflects the total cholesterol content of all apoB-lipoproteins ⁴⁴. Here the apoB₁₀₀ lineage dominates and is secreted by the liver and metabolised systemically, where VLDL undergoes lipolysis (via LpL) to IDL and LDL, while HDL transfers (via CETP) cholesteryl esters (CEs), thereby generating increasingly triglyceride-poor and cholesterol-rich particles. LDL is the major cholesterol carrier and has a long plasma residence (in the order of days) before being endocytosed via the LDL receptor (LDLR) and largely cleared by the liver ⁴⁵, where it subserves (indirect) reverse cholesterol transport; i.e. ~70% of HDL-CEs are transferred to VLDL/LDL before hepatic uptake ⁴⁶. However, the majority of peripheral cholesterol clearance may occur more rapidly via direct uptake of HDL-derived free cholesterol ^47,48.

Conversely, in early atherogenesis lipids and lipoproteins accumulate in arterial intima at susceptible sites (e.g. branches and bifurcations) ^49–51, and prior to macrophages ³⁷. These sites are typically exposed to turbulent blood flow and shear stress and often exhibit increased lipoprotein permeability and/or retention ⁵². 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 ^37,49,50, while exposure to various enzymes and oxidants promote modifications, ultimately resulting in lipoprotein aggregation, fusion and cholesterol crystallisation ^36,53,54. Notably, among plasma lipoproteins small-dense LDL exhibits various qualities which may render it particularly atherogenic ⁵⁵, albeit while carrying a smaller cholesterol load ⁴²; indeed when controlling for particle count, large and small LDL similarly relate to CVD risk ⁵⁶. 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 ⁵⁷ and inflammasome activation ³⁶. This reflects a dichotomy: foam cells remove and digest harmful extracellular deposits liberating free cholesterol, with potential for efflux to HDL and out of arteries, but when overloaded can themselves be a site of crystallisation, inflammation and cytotoxicity ^51,58. Cholesterol crystals may also form early in experimental atherosclerosis (i.e. pre-macrophage) via endothelial cells, which also process LDL but seem less able to upregulate efflux ⁵⁹. Such crystals can impair the endothelial barrier (to leukocytes) ⁵⁹, and activate endothelial ⁶⁰ and macrophage NLRP3 inflammasomes—a pathway required for atherogenesis, and which releases cytokines recruiting more immune cells ⁶¹.

Beyond the arterial wall, plasma lipids also modulate important extravascular pathways. For instance, hypercholesterolemia suppresses and lipid-lowering restores bone marrow-derived endothelial progenitor cells (EPCs), which mediate vascular repair ^62,63. Hypercholesterolemia also acts on bone marrow and peripheral cells to favour platelet biogenesis and activation ⁶⁴, as well as monocyte skewing and activation ⁶⁵, thereby priming the precursor to tissue macrophage foam cells ⁶⁶. Furthermore, circulating monocytes can also internalise CE-rich lipoproteins, become foamy and inflammatory, and subsequently infiltrate nascent lesions ⁶⁷.

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. Notably, 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 which was also partially mediated via plasma cholesterol ⁶⁸. 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-source PUFAs (i.e. C18:2/3) lower it, as tested in 100s of metabolic ward studies ¹⁵ and formularised since the 1950s ⁶⁹. This dichotomy extends to low carb/keto diets ^70–73; although more extreme hypercholesterolemia may relate to leanness and greater TG/VLDL turnover ⁷⁴ (even when favouring MUFAs ⁷⁵). The effect of SFAs may be accentuated by dietary cholesterol ^76,77, attenuated in the case of cheese (vs. butter) ⁷⁸ and modulated by APOE genotype (i.e. E4 ^79,80 and -219G/T ⁸¹). SFA-rich diets similarly increase apoB ^78,82–85 (meta-regression in ¹⁶); more specifically, dairy fat/SFAs can increase all VLDL–LDL particles (vs. seed oils/n-6 PUFAs) ^82,86, large LDL (vs. MUFAs) ⁸⁴, or in people with pattern B, medium–small LDL particles (vs. MUFAs) ⁸⁵. Mechanistically, human and animal studies suggest SFA-rich diets suppress LDL catabolism ⁸⁷ and LDLR expression ^82,83, thereby prolonging LDL residence in blood ⁸⁸, but in a manner dependent upon dietary cholesterol and UFAs ^89,90. However, even mice made prone to atherosclerosis via genetically impaired (hepatic) uptake exhibit differential effects. For instance, in APOE^–/– or LDLR^–/– mice a coconut oil/SFA-rich diet (vs. fish oil/n-3 PUFAs) greatly increased arterial uptake of whole LDL and CEs, paralleling macrophage LpL and lesion development ^91–93, while in LDLR^–/– mice replacing SFAs with MUFAs (i.e. dairy fat with extra-virgin olive oil and nuts, respectively) decreased circulating foamy-inflammatory monocytes and atherosclerosis ⁹⁴.

SFAs may also affect atherogenesis via modulation of triglyceride-rich lipoproteins (TRLs) ⁹⁵, which constitute non-LDL/HDL-associated ‘remnant cholesterol’ ⁹⁶. The effect of different dietary fats on postprandial lipemia in humans so far seems equivocal (reviewed in ⁹⁷). However, in healthy adults high fat/SFA meals (vs. low fat) can induce foamy-activated monocytes in association with postprandial TRLs ^98–101, while in vitro SFAs (vs. MUFAs and PUFAs) induced greater monocyte lipid droplet formation ¹⁰⁰. As intestine-derived apoB₄₈-chylomicrons are depleted of their triglycerides they become remnant particles, which are rich in cholesterol and associated with atherogenesis (i.e. analogous to conversion of VLDL to LDL). In preclinical studies enrichment of chylomicron remnants with SFAs (vs. various UFAs) reduces hepatic uptake but induces macrophage lipid accumulation ¹⁰². Further, in humans SFA-rich meals (i.e. palm oil and cocoa butter vs. various UFAs) induced apoE expression in TRLs which in vitro increased hepatocyte LDLR binding resulting in competitive inhibition of LDL uptake ¹⁰³; this effect was even greater in those with an APOE4 allele, which is itself associated with both Alzheimer’s and CVD ⁸⁰. Notably, in mice a cocoa butter/SFA-rich diet (not MUFAs or fish oil/n-3 PUFAs) also increased amyloid-beta (Aβ) in gut enterocytes and plasma TRLs ^104,105, and induced blood–brain barrier (BBB) dysfunction and Aβ transport to the brain ¹⁰⁶; potentially underlying the association between SFA intake and Alzheimer’s (reviewed in ¹⁰⁷). Reciprocally, dementia and plasma Aβ₄₀ are associated with CVD ¹⁰⁸, while Aβ_40/42 binding to native or modified LDL enhanced foam cell formation in vitro ¹⁰⁹.

It’s generally thought that lipoproteins must first be modified in some way to promote atherogenesis. However, this may not be strictly true since atherogenic concentrations of native LDL in vitro can induce endothelial dysfunction ¹¹⁰ and permeability ¹¹¹ (e.g. reviews ^58,63), which was recently linked to LDL uptake and cholesterol crystallisation ⁵⁹. Further, high levels of native LDL can dose-dependently induce macrophage foam cell formation in a non-saturable manner via receptor-independent fluid-phase pinocytosis ¹¹² and selective CE uptake ¹¹³; as well as CCL20 secretion by vascular smooth muscle cells, causing lymphocyte migration ¹¹⁴. Regardless, lipoprotein modifications have important amplifying and auxiliary effects…

Oxidation

Oxidised LDL (oxLDL) is present in plaques and plasma where it’s associated with CVD ^115,116, although not always independently of apoB (e.g. CHD ¹¹⁷ and MetS ¹¹⁸), likely due to 4E6 antibody cross-reactivity ¹¹⁹. 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 ¹¹⁹. OxLDL normally represents a very small fraction of plasma LDL ¹²⁰ and increases preceding progression and regression of experimental atherosclerosis, suggesting exchange with plaque ¹²¹; similar to oxPL–apoB ¹¹⁹. 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 ⁵⁸, CCL20 ¹¹⁴, EPCs ⁶³ and HSPCs ¹²²) and induces macrophage uptake via scavenger receptors ¹¹⁶. Here it induces lipid droplets, which may be limited by defective lysosomal processing ¹¹², and also lysosomal crystals and NLRP3 activation ^61,123. As the major transition metal in vivo, iron (Fe²⁺) dysregulation may particularly promote oxidation during plaque haemolysis ¹²⁴. Moreover, many studies also report that human plaques have increased expression of inducible nitric oxide synthase (iNOS) ^125–129 and myeloperoxidase (MPO) ¹³⁰ (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) ^131,132, implicating immune-dependant redox modifications ¹³³. These oxidations are not blocked by serum (unlike copper oxidation) and resulting NO₂–LDL stimulates macrophage uptake and cholesterol loading via scavenger receptor CD36 ^134,135. 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 ⁵⁷. Indeed LDL aggregation greatly increases macrophage uptake by receptor-independent endocytosis ^57,113 and CE accumulation beyond native or oxLDL ^136,137. Further, mildly oxidised LDL inhibits native LDL-induced foam cell formation ¹³⁸, although such particles tend to aggregate ⁵³ and still induce macrophage crystals ^61,123, so may contribute in this way ⁵⁷.

Of lipids PUFAs are particularly susceptible to oxidation, of which linolenic acid (LA; C18:2, n-6) is the major form in LDL and plaque CEs. A substantial proportion of plaque CE-LA is oxidised ¹³⁹, despite normal levels of the major lipophilic antioxidant α-tocopherol (aka. vitamin E) ^140,141; i.e. contrasting typical conditions in vitro and consistent with failed antioxidant trials ¹³³. Further, under strong copper-oxidising conditions α-tocopherol acts as a chain-breaking antioxidant and underlies the lipid oxidation lag phase, whereas under more mildly oxidising conditions the α-tocopherol radical can initiate lipid oxidation ¹⁴², or when there are insufficient regenerative co-antioxidants (e.g. CoQ₁₀ and carotenoids) ¹⁴³; and α-tocopherol does not block iNOS/MPO-derived oxidants ¹⁴⁰. Importantly, human aortic lesions from early to end-stage disease had accumulation of non-oxidised cholesterol and CEs before their oxidised derivatives ¹⁴⁴, consistent with the ‘response-to-retention’ model ^49,50. More recent high-resolution imaging of advanced carotid plaques has further revealed that oxidised CEs co-localise with sphingomyelin in the necrotic core ¹⁴⁵. Of potential relevance, oxidation of LDL-c generates 7-ketocholesterol, which in macrophages inhibits lysosomal sphingomyelinase (SMase) causing accumulation of sphingomyelin–cholesterol particles ¹⁴⁶, and also dose-dependently induces cholesterol crystals ¹⁴⁷.

Regarding diet, in various short-term trials a lower total fat/SFA or higher olive oil/MUFA intake can lower LDL susceptibility to copper oxidation and monocyte adhesion in vitro, correlating the LDL oleate/linoleate (i.e. MUFA/PUFA) ratio ^148–150; conversely, these effects increased most with a mixed n-6/n-3 PUFA diet ¹⁴⁸. However, the relevance of this is questionable since it doesn’t parallel favourable associations between PUFAs and hard outcomes, or consider changes to pathways in vivo which initiate oxidation (e.g. iron and inflammation) and increase apoB-lipoproteins (i.e. total substrate) ¹⁵⁰. Importantly, the omega-3 content of advanced carotid plaques is increased by supplementation and correlates greater stability and lower inflammation, consistent with anti-inflammatory effects ^151,152. Furthermore, the food matrix is also important. For instance, in healthy adults a PUFA-rich walnut meal (i.e. 59g fat, 42g PUFA, 34g LA) increased blood antioxidants and lowered oxLDL at 2 hours (i.e. Fig. 1) ¹⁵³, while longer trials show enrichment of PUFAs with preservation of oxidation status ^154,155, alongside many other cardio-protective effects (reviewed in ¹⁵⁶). Conversely, red meat and heme-iron intake are associated with CVD ^157–160 and can promote lipid oxidation during digestion ¹⁶¹. For instance, in humans and mice red meat ingestion induced postprandial plasma lipid oxidation (i.e. 3x MDA) and corresponding LDL modification, which was greatly inhibited by polyphenols ^162,163; and in gastric models olive oil/MUFAs inhibited red meat/iron-induced lipid peroxidation, opposite to fish oil/n-3 PUFAs ¹⁶⁴. Notably, in a unique RCT comparing oxidised vs. high quality fish oil, only the latter lowered apoB-lipoproteins ¹⁶⁵.

In preclinical studies HDL has many anti-atherogenic activities, while several human studies report that cholesterol efflux capacity from macrophage to HDL predicts CVD risk better than HDL-c ⁴⁶. Conversely, apoA-I/HDL isolated from human plaques is lipid-poor and pro-inflammatory as a result of various modifications ¹⁶⁶, including chlorination, as above. In particular, the first stage of reverse cholesterol transport involves ABCA1-dependent efflux to apoA-I forming nascent HDL ⁴⁶, which is inactivated by MPO ^132,167. These pathways may be modulated by dietary fats. For instance, healthy adults (n=122) with higher insulin resistance or SFA intake (>10% kcal) had lower ABCA1-dependent efflux (independent of HDL-c) ¹⁶⁸, while in mice SFA-rich diets lowered liver­–faeces cholesterol transport and enriched HDL with hepatic-derived acute-phase (inflammatory) proteins (i.e. palm oil/SFAs vs. sunflower oil/MUFAs) ¹⁶⁸, and impaired HDL antioxidant activity (i.e. dairy fat/SFAs vs. soybean oil/n-6 PUFAs) ¹⁶⁹. Accordingly, in young healthy adults a single coconut oil/SFA-rich meal (vs. safflower oil/n-6 PUFAs) suppressed HDL anti-inflammatory activity and flow-mediated dilation (FMD) ¹⁷⁰. Further, 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 ⁹⁸; and in vitro cow milk fatty acids (i.e. oleate or palmitate) induced MPO release by monocytes and uptake by porcine arteries ⁹⁸.

Sphingolipids

Alongside cholesterol, another significant component of plaque is sphingolipids ³³. Here various sphingomyelins and ceramides are present and associated with plaque inflammation and apoptosis ¹⁷¹, while serum ceramides (esp. Cer16:0, Cer18:0 and Cer24:1) predict CVD risk independent of conventional risk factors (incl. apoB) ¹⁷². Moreover, 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 ¹⁷³). Serum ceramides are particularly elevated in obesity and type-2 diabetes (T2D)—itself a major CVD risk factor ¹⁷². In particular, LDL ceramides were specifically elevated in T2D (not obesity) and in preclinical models induce macrophage activation and muscle insulin resistance ¹⁷⁴, which involves mitochondrial dysfunction (reviewed in ¹⁷⁵). Such insulin resistance promotes hyperglycaemia, which is also associated with CVD ¹⁷⁶, while a 1-year RCT with insulin-stimulating drugs induced regression of carotid intima-media thickness (cIMT) in relation to postprandial glucose ¹⁷⁷. Note, even in the nondiabetic PESA cohort HbA1c (i.e. monthly glucose control) independently correlated the presence of subclinical atherosclerosis ³¹. Accordingly, hyperglycaemia can induce non-specific glycation, oxidative-inflammatory activity and endothelial dysfunction ¹⁷⁶; in particular, glycation of LDL increases arterial proteoglycan binding ^178,179. Insulin itself also promotes hepatic apoB metabolism ¹⁸⁰ and may affect many atherogenic cells ^181,182.

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 ¹⁸³ and sphingolipids/ceramides ¹⁸⁴ correlate insulin resistance, while SFA intake was increased in T2D ¹⁸³. Accordingly, in some trials on healthy adults a higher palmitate/SFA intake (vs. oleate/MUFA) for 2–3 weeks ^185,186, or as a single bolus ¹⁸⁷, induced blood/muscle sphingolipids/ceramides and suppressed glucose metabolism and insulin sensitivity. Further, in trials on overweight adults over-feeding SFAs from mixed sources (vs. UFAs or sugars) for 3 weeks ¹⁸⁸, or palm oil/SFAs (vs. sunflower oil/n-6 PUFAs) for 8 weeks ¹⁸⁹, increases multiple plasma/LDL sphingolipid species (opposite to PUFAs), paralleling insulin resistance and liver fat (i.e. intrahepatic triglycerides). Conversely, 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 ¹⁹⁰. Notably, in mice and human cells palm oil/SFAs (vs. olive oil/MUFAs) were also reported to induce intestinal insulin resistance via ceramide, thereby impairing the ability of insulin to inhibit triglyceride secretion and linking to postprandial hypertriglyceridemia ¹⁹¹.

Besides glucose-insulin homeostasis, sphingolipids may have more direct effects on atherogenesis. For instance, LDL can deliver ceramide to endothelial cells ¹⁹², where it can mediate apoptosis ¹⁹², suppress nitric oxide (i.e. eNOS) ¹⁹³ and increase the uptake and retention of oxLDL ¹⁹⁴. 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 ⁵³. 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) ¹⁹⁵. LDL aggregation was also related to the surface/core lipidome (esp. ↑ sphingolipids/ceramides vs. phospholipids) which was favourably modified by a ‘Healthy Nordic diet’ (incl. ↑ PUFAs; Fig. S5) or lipid-lowering drug (i.e. PCSK9 inhibition) ¹⁹⁵. 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 ¹⁹⁶. A further study including liver biopsies reported that LDL aggregation and lipid composition correlates the liver lipidome, implicating hepatic sphingolipid metabolism in LDL composition ¹⁹⁷. Also, in LDLR^–/– mice a dairy fat/SFA and cholesterol-rich diet induced macrophage S-SMase which acts on serum LDL to increase ceramide and susceptibility to aggregation and oxidation ¹⁹⁸.

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 ¹⁹⁹. Nowadays many studies support a link with cardiometabolic disease and athero-thrombosis (e.g. reviews ^200–202). LPS is the canonical ligand for toll-like receptor 4 (TLR4), which stimulates innate immunity and primes the NLRP3 pathway ^36,61,123; although depending on source/structure, it can act as an agonist or antagonist (e.g. E. coli and Bacteroides, respectively) ²⁰³. In mouse models of endotoxemia, platelet TLR4 triggers neutrophil extracellular traps (NETs) to ensnare bacteria in liver sinusoids and pulmonary capillaries ^204,205, but which also promote monocyte recruitment and aortic atherosclerosis ^206,207, and increase carotid plaque MPO and instability ²⁰⁸, consistent with human samples ^207,208. In particular, E. coli-LPS was present in human carotid plaques (esp. necrotic core; Fig. 1A) and associated with enlarged macrophages, while plaque levels tested in vitro induced monocyte NADPH oxidase 2 (Nox2) and oxLDL, implicating TLR4-mediated oxidative stress ²⁰⁹. Further, carotid LPS correlated plasma LPS (r=0.668), which correlated soluble TLR4 and serum zonulin (a marker of intestinal permeability) ²⁰⁹; with similar blood marker relationships reported in other populations ^210–212. 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) ^213–215. Butyrate is the archetypal beneficial short-chain fatty acid (SCFA) and ameliorates atherosclerosis in APOE^–/– mice by lowering gut permeability and endotoxemia ²¹⁶ and inducing ABCA1-dependent cholesterol efflux ²¹⁷. Several human studies also find depletion of Bacteroides spp. ^213,218,219, which when administered to APOE^–/– mice also lowered atherosclerosis and gut/blood LPS ²¹⁸.

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) ²²⁰. In blood both LPS and Gram-positive lipoteichoic acid (LTA), as exogenous lipid-based molecules, are largely bound to lipoproteins ²²¹, transferred to LDL (via PLTP) ^222,223 and removed predominantly via the hepatic LDLR (in humans) ^224,225, suggesting dietary fats may also differentially regulate LPS clearance. A single LDL particle can bind many LPS molecules with only minor changes to its composition ²²⁶. While such lipoprotein binding sequesters the lipid A region of LPS within the phospholipid monolayer and hepatic uptake is apparently non-toxic ^224,225, chylomicrons and LDL may still carry bioactive LPS across other tissues to elicit inflammation (e.g. lymph nodes ²²⁷, endothelium ²²⁸, adipose ²²⁹ and brain ²³⁰). In particular, LDL–LPS complexes formed in vitro acquire a negative charge and have increased binding and accumulation in arterial wall and macrophages ²³¹. LPS also induces smooth muscle cell synthesis of elongated proteoglycans (equal to traditional agonists) ²³², increases LDL susceptibility to oxidation (by copper, endothelial and smooth muscle cells) ²³³, and stimulates macrophage oxLDL uptake and foam cell formation ²³⁴; while at concentrations in CAD, LPS and indoxyl sulfate (a microbial metabolite of tryptophan) exhibited co-toxicity on endothelial cells ²¹⁹. Moreover, in humans and rodents LPS impairs total (i.e. macrophage–faeces) reverse cholesterol transport at multiple steps ²³⁵; in part via induction of MPO/SAA ²³⁶ and suppression of ABCA1 ²³⁷.

Regarding dietary fats, in a 3-week trial on overweight adults (similar to those above ^188,196), over-feeding SFAs increased faecal Gram-negative Proteobacteria, while UFAs increased butyrate-producing bacteria (i.e. Lachnospira, Roseburia and Ruminococcaceae spp.) ²³⁸. Similarly, in a double-blind crossover trial, replacement of butter/SFAs with margarine/PUFAs (i.e. seed oils/n-6 PUFAs) for just 3 days induced Lachnospiraceae and Bifidobacteria, with the former negatively correlating total cholesterol (r=­–0.511) ²³⁹. While not generally altered in CVD ²¹³, SFA-rich diets also associate with elevated bile-resistant, sulfide-producing genera (i.e. esp. Bilophila; in faeces ^238,240 and mucosa ²⁴¹), which in mice results from increased secretion of taurine-conjugated bile acids ²⁴² and lowers colonic butyrate and barrier function ^242–244. Moreover, in humans SFA-rich meals can increase postprandial plasma LPS (systematic review ¹⁸). For instance, dairy fat (vs. carbs, MUFAs and n-3 PUFAs) increased postprandial LPS, paralleling immune cell activation and endothelial adhesion markers ^245–247; an effect quicker in obesity ²⁴⁷ and present after 12 weeks of a SFA-rich diet ²⁴⁶. A porridge-based meal with coconut oil/SFAs (vs. fish oil/n-3 PUFAs) also increased postprandial LPS, but not serum cytokines ²⁴⁸. 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; while plasma LPS concentrations incubated with platelets in vitro induced TLR4-dependent Nox2 and oxLDL ²⁴⁹. Accordingly, in preclinical studies fat-induced LPS absorption occurs in the small intestine via transcellular chylomicron and portal vein pathways (i.e. lymph and liver, respectively) ^227,250. Short-term controlled-feeding crossover trials in healthy adults also report that lowering typical dietary SFA/MUFA ratios lowers LPS-induced cytokine secretion ^251,252, suggesting SFA-rich diets may further increase LPS sensitivity.

In human and experimental fatty liver, hepatocyte LPS is also increased, associated with immune-inflammatory markers ²⁵³ and implicated in pathogenesis (e.g. reviewed in ²⁵⁴). Various short-term trials show that SFAs in particular can induce liver fat, under isocaloric (i.e. butter/SFAs vs. sunflower oil/n-6 PUFAs ²⁵⁵) or hypercaloric conditions (i.e. SFAs vs. UFAs ^189,256 and sugars ^188,238); and in association with adipose lipolysis and inflammation, endotoxemia (i.e. LBP/CD14 ratio) and baseline levels of faecal Bilophila ^188,238. Accordingly, in mice Bilophila wadsworthia aggravates dairy fat/SFA-induced metabolic dysfunctions and steatosis, while suppressing microbial butyrate and promoting LPS biosynthesis and translocation ²⁴³. Also, in healthy adults a single bolus of palm oil/SFAs acutely induced whole-body/muscle/liver insulin resistance and intrahepatic triglycerides, where a parallel mouse study revealed transcriptomic evidence of hepatic LPS/TLR signalling ²⁵⁷. Notably, the intestine secretes HDL₃ into the portal blood which can bind LPS and restrain high fat diet-induced liver injury and fat storage ²⁵⁸. In humans experimental endotoxemia induces peripheral inflammation, oxidative stress and lipolysis (i.e. free fatty acids), the latter 2 of which were particularly inhibited by co-infusion of insulin ²⁵⁹. Endotoxemia also induces ceramides in VLDL and LDL ²⁶⁰, which in rodents is accompanied by activation of S-SMase in serum and de novo sphingolipid biosynthesis (i.e. SPT) in liver ^260,261. Therefore LPS may mediate some of the changes induced by SFAs above, and consequently differential effects to UFAs. Notably, in other postprandial trials cream-induced liver fat was attenuated by co-administration of 50g glucose (consistent with the ability of insulin to inhibit lipolysis) but not fructose ²⁶², which itself may also be capable of inducing endotoxemia ²⁶³, DNL and ceramides ²⁶⁴.

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. Generally, the plasma membrane exists 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 ²⁶⁵. Such rafts may serve as functional platforms to assemble proteins subserving cell signalling and endocytosis ²⁶⁶, which can be modulated by exogenous lipids. In particular, free cholesterol in the lipoprotein monolayer is in equilibrium exchange with cell membranes ²⁶⁷, while VLDL and LDL were reported to preferentially interact with membrane raft regions, consistent with the high affinity of apoB₁₀₀ for cholesterol (and contrasting triglyceride-rich chylomicrons) ²⁶⁸. Further, the hepatic LDLR is associated with both clathrin and caveolae-rich membrane regions ²⁶⁹ (which correspond to non-raft and raft regions, respectively ²⁶⁶), and treatment with LDL or cholesterol induced translocation to caveolae coinciding with reduced LDL uptake ²⁶⁹. In addition, internalised LDL-c travels from lysosomes to plasma membrane first, then endoplasmic reticulum (ER) regulatory domains, which can suppress SREBP and thereby cholesterol uptake and synthesis, to maintain homeostasis ²⁷⁰. While in the other direction, the ABCA1 transporter may associate with cholesterol-rich lipid rafts ²⁷¹ to mediate efflux to apoA-I/HDL ²⁷²; indeed the composition of nascent HDL resembles lipid rafts ²⁷². 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 plasma membrane incorporation and compensatory induction of saturated lipids and cholesterol to maintain biophysical homeostasis ²⁷³. Conversely, SFAs can induce saturated glycerolipids in ER and solid phase (i.e. solid-order, S_o) microdomains, in a manner correlating SFA chain length and offset by UFAs ²⁷⁴. In rodent studies SFAs (i.e. C12–16:0) are also relatively poor substrates for ACAT-dependent cholesterol esterification, which may increase free cholesterol in the ER to inhibit SREBP-dependent LDLR expression, thereby increasing plasma cholesterol ⁸⁹, whereas UFAs (C18:1 > C18:2) stimulate greater hepatic CE synthesis and secretion, as well as LDLR activity, thereby increasing plasma cholesterol turnover ²⁷⁵. In addition, human plasma CEs are especially rich in n-6 PUFAs (i.e. C18:2 > C18:1 > C16:0), even on a high SFA diet ⁷¹, consistent with the phospholipid fatty acid preference of LCAT, which mediates esterification in plasma lipoproteins, especially HDL. Accordingly, in humans and rats dietary fat saturation modulates LCAT activity (i.e. n-6 PUFAs > MUFAs > SFAs), which itself correlates plasma linoleate and inversely with oleate ^276–278, and in one case also total cholesterol ²⁷⁸. Further, LCAT overexpression in transgenic rabbits elevates HDL-c and lowers LDL-c via the LDLR ²⁷⁹. In this respect, HDL-CEs are transferred to VLDL/LDL ⁴⁶ and UFAs 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) ²⁸⁰. In monkeys corn oil/n-6 PUFAs (vs. coconut oil/SFAs) increased LDL uptake by PBMCs which correlated membrane fluidity and lower plasma cholesterol ²⁸¹; and enrichment of hepatocytes in various fatty acids (i.e. n-6 PUFAs > MUFAs > SFAs) affected LDL binding/metabolism and membrane fluidity in a highly correlated manner, without altering total or esterified cholesterol ²⁸². Similarly, in hamsters decreasing the dietary fat saturation also increased HDL–liver membrane binding ²⁸³.

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 ⁵⁸). 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 ²⁸⁴. As above, LDL can also deliver ceramide to endothelial cells ¹⁹², where endogenous ceramide promotes the uptake and retention of oxLDL via regulation of transcytosis-related and raft-associated proteins, including LOX-1 ¹⁹⁴. Further, conditions of hypercholesterolemia and 7-ketocholesterol induce endothelial A-SMase/ceramide-dependent membrane raft redox signalling platforms linked to NLRP3 activation ²⁸⁵. Notably, electronegative LDL also possesses intrinsic SMase activity associated with apoB₁₀₀ serine O-glycosylation ²⁸⁶; this may be outward-facing so as to engage plasma membrane sphingomyelin, generating ceramide-based microdomains and endocytic vesicles ^287,288. Moreover, arterial SMase can hydrolyse sphingomyelin within lipoproteins themselves generating ceramide-rich domains, which may act as nonpolar spots promoting aggregation via hydrophobic interaction ⁵³, as well as displacement and release of cholesterol to neighbouring vesicles ²⁸⁹. Atherosclerotic plaques were also reported to contain membranes enriched in free cholesterol and crystalline domains ⁵⁸. In preclinical studies plaque crystals co-associated with cholesterol microdomains ²⁹⁰, which can be shed from macrophage membranes ²⁹¹. Rapid loading of macrophages via phagocytosis of large lipid droplets induces lysosomal free cholesterol and extracellular crystals ⁵¹; 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 ^292,293. Further, lipid oxidation induces crystalline domains in model membranes under conditions of hyperglycaemia, which can be inhibited by n-3 PUFAs, especially EPA ²⁹⁴. 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 ²⁹⁵.

Statins have well-known anti-inflammatory effects. In particular, 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; pushing postprandial MNC TLR2/4 expression even below baseline (i.e. Fig. 3G/H) ²⁴⁷. In macrophages TLR4 activation requires cholesterol biosynthesis (via FASN) to enter lipid rafts ²⁹⁶, while ABCA1-dependent cholesterol efflux suppresses raft-associated TLR/inflammatory signalling in macrophages ^297,298 and endothelial cells ²⁹⁹. Furthermore, in various human cohorts total plasma cholesterol was independently associated with NK cell cytotoxicity, which was lowered by statins or MβCD in vitro ³⁰⁰; and LDL-c correlated a haematopoietic monocyte skewing (vs. granulocytes) in blood ⁶⁵ and proinflammatory macrophage phenotype in adipose ³⁰¹, which were suppressed by statins in vivo. 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 (i.e. the common β subunit of GM-CSF and IL-3 receptors) between raft and non-raft fractions, respectively ³⁰².

Importantly, lipid rafts are involved in endocytosis ²⁶⁶ and represent a common entry point for many viral, bacterial and fungal pathogens ^303–305. For instance, in the colon butyrate may inhibit enteric pathogen invasion via depletion of cholesterol and increased membrane fluidity ³⁰⁶. Similarly, in porcine ileum samples SFA-induced LPS permeability was abrogated by MβCD, implicating lipid rafts ³⁰⁷. Intriguingly, intestinal enterocytes were reported to phagocytose and translocate E. coli/LPS via TLR4 ³⁰⁸, which in macrophages is recruited to lipid rafts in response LPS, and also SFAs, but inhibited by n-3 PUFAs ³⁰⁹ (i.e. paralleling effects on postprandial LPS ^248,307). Accordingly, bacterial LPS and lipopeptides are acylated with chains of SFAs which engage TLR4 and TLR2, respectively ³⁰⁹. For instance, in E. coli LPS the lipid A region is typically hexa-acylated with C14/12 SFAs which interact with MD2 lipid domains inducing TLR4 dimerization, whereas hypo-acylation or incorporation of UFAs result in antagonist activity ^309,310. Indeed total gut LPS silences TLR signalling due to hypo-acylated lipid A in Bacteroidales ²⁰³. Consequently, plasma free SFAs have been suggested to mediate LPS-associated postprandial inflammation ³¹¹. However, palmitate may not act as a direct ligand for TLR4, but facilitate activation via metabolic reprogramming ³¹². Of potential relevance, LPS and oxidative stress-induced TLR4 complex formation also requires A-SMase-derived ceramide ^313,314 (which has structural similarities to lipid A ³¹⁵), while palmitate augments LPS inflammatory responses via SMase and de novo ceramide synthesis ^316–319. Free SFAs (i.e. palmitate and stearate) can also activate macrophage NLRP3 inflammasomes via flux into phosphatidylcholine ³²⁰ and crystallisation ³²¹, which are offset by UFAs.

Ecology

Evidence of atherosclerosis has been reported in ancient humans spanning 4000 years and from diverse locations suggesting a basic predisposition ³²²; 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 ³². These populations may have been exposed to various enduring risk factors, including diet, smoke and infections, although this remains speculative ³²³. However, there are some notable examples of extant pre-industrial people with divergent health outcomes, suggesting post-industrial changes are also important ^324,325. 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 ³²⁶; as well as atrial fibrillation ³²⁷, age-related brain atrophy ³²⁸ and dementia ³²⁹.

Lipids likely play a fundamental role in our susceptibility to CVD. Indeed many other mammals are relatively resistant to atherosclerosis and their use as experimental models requires ‘humanisation’ of the lipid profile, by genetically increasing apoB-lipoproteins (e.g. APOE^–/– or LDLR^–/– mice) and/or feeding a high cholesterol/high fat diet ³³⁰. Such experimental atherosclerosis requires ACAT2 (aka. SOAT2), which is expressed in intestine and liver where it synthesises MUFA-rich CEs for apoB-lipoproteins ^331,332, and mediates LDL proteoglycan binding ³³³ and aggregation ¹⁹⁵. However, in humans a high oleic oil diet enriched CEs but lowered LDL biglycan binding ³³³, suggesting further differences; of note, other mammals may express higher ACAT2 ³³⁴ and are often fed higher dietary cholesterol ^89,333. Experimental atherosclerosis also requires NLRP3, which links microbial and/or sterile cell stress to innate immunity ⁶¹. Today many infections are associated with atherosclerosis and the acute-phase response encompasses mutual changes supporting immunometabolism and defence reminiscent of those implicated in CVD ^335,336. For instance, infections/inflammation induce macrophage aerobic glycolysis and accumulation of lipid droplets with antimicrobial activity ^337,338, while also modulating systemic insulin sensitivity (i.e. glucose metabolism) ^339,340, lipid metabolism (e.g. lipolysis, cholesterol and sphingolipids) and lipoprotein modifications (e.g. oxLDL) ³³⁶. Induction of vascular retention and LDL oxidation might even support bacterial sequestration (e.g. LPS­–TLR4 ^204,232 and M. tuberculosis ⁵⁰) and phagocyte clearance (e.g. LPS ^233,234). These mechanisms could therefore support acute survival, while persistent stimulation exacerbates vascular disease hastening late-life mortality ³³⁶, as a form of antagonistic pleiotropy. Indeed while CVD is currently the leading cause of death, ancestrally it was likely injury and infections, 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 ³⁴¹. The specific composition of LPS varies between bacteria and is modulated by environmental factors ³⁴¹. In pathogenic bacteria the lipid A region is typically hexa-acylated with SFAs ³¹⁰, 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 ³⁴². 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 ³⁴³. In mice SFA-rich diets (i.e. C12/16:0) exacerbated central autoimmunity by increasing Th1/17 activity via the small intestine ^344,345. Th17 cells express particularly high levels of TLR4, and LPS directly induces Th17 differentiation in vitro ³⁴⁶. 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 ³⁴⁷. 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 ³⁴⁸. This was consistent with SFA induction of innate immune memory (i.e. ‘trained immunity’), itself another double-edged sword ³⁴⁹. 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 ⁶⁵. Dietary cholesterol may also affect the pathophysiology of infectious and autoimmune disease (reviewed in ³⁵⁰).

The ability of exogenous lipids to differentially regulate endogenous lipids and physiology may arise from several key factors. Foremost, the dependence of cell membrane biophysics and 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 ^274,320,321. Accordingly, the ER-associated enzyme SCD1 mediates endogenous desaturation of SFAs to MUFAs and limits experimental atherosclerosis and macrophage TLR4 hypersensitivity, potentially via suppression of L_o microdomains, albeit at the cost of obesity-related metabolic disease ³⁵¹. 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 to steroidogenic and hepatic tissues, so excess must be esterified for safe storage or undergo efflux and 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 ^89,275 and LCAT ^276–278), 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 ³³⁶.

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 ³⁵². An increase in dietary cholesterol and fats from animals (i.e. land and marine) and plants (esp. nuts/seeds) may have modulated plasma cholesterol in relation to the SFA/PUFA ratio ^{76,77,84,89,90}. 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) ³⁵² and animal food aversion during early pregnancy (i.e. morning sickness), a relatively immune-suppressed period ^353,354. The advent of cooking would support sterilisation and may be reinforced by the appealing sensory qualities of advanced glycation end products (AGEs) ³⁵⁵, albeit at the potential cost of cardiometabolic dysregulation, as seen in modern humans ³⁵⁶. Despite these factors native populations often exhibit relative cardiometabolic health ^324,325. In particular, our genus and species emerged in Africa, where remaining hunter-gatherer exemplars such as the Hadza of Northern Tanzania maintain a low body weight, blood pressure and plasma lipids throughout life, with a relatively low fat intake (i.e. median ~18% kcals) from plants and lean meats ³⁵². Similarly, Tsimane vascular health is accompanied by low plasma/LDL cholesterol ³²⁶, with a very low fat and SFA intake (i.e. men: 15.1% and 3.7% kcals, respectively) from a plant-dominant diet with moderate fish/meat ³⁵⁷. Furthermore, in this energy-limited and pathogenically diverse context, the ancestral APOE4 allele is actually associated with better cognition in those infected with parasites ³⁵⁸, 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 ³⁵⁹, 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) had higher LDL-c and apoB, but which was the most responsive to lowering when replacing SFAs with low GI carbs, not MUFAs ⁷⁹.

Industrialization has led to another nutrition transition, wherein major SFA sources are now grain-fed meats, which are richer in total fat and specific fatty acids ³⁶⁰, along with concentrated animal/dairy and tropical fats (as used in SFA trials herein), in the context of a diet high in ultra-processed foods/calories, with a high n-6/3 PUFA ratio, and low in micro-/phytonutrients, implicating evolutionary mismatch in SFA-associated diseases ³²⁵. Indeed many nutritional and physiological factors may modulate the effects of dietary SFAs today. For instance, SFA-induced postprandial inflammation may especially occur in obesity ^247,249,361, but be blunted by lipid-lowering therapy ²⁴⁷ or co-ingestion of phytochemicals (e.g. polyphenols ³⁶², spices ³⁶³ and fibre ³⁶⁴), which can also accompany UFAs (e.g. olive oil and nuts). Long-chain n-3 PUFA status may also be important via anti-LPS/TLR effects ^248,307,309; cellular levels are also highly responsive to diet at the expense of n-6 PUFAs ³⁶⁵ (see supplement). SFA-induced liver fat may especially be promoted by overfeeding ^{188,189,238,256}, and exacerbated by excess fructose ²⁶². Conversely, low carb diets increase fat oxidation and may mitigate the differential effects of SFAs (vs. UFAs) on insulin sensitivity and inflammation, but not cholesterol and ketones ^70–73. Further, ketosis may have inherent anti-inflammatory effects ³⁶⁶, and a 3-day isocaloric ketogenic diet suppressed LPS/palmitate-induced inflammasome activation in macrophages ex vivo ³⁶⁷.

While many factors may contribute to atherogenesis, as a condition of arterial lipid accumulation, a lipid-threshold may ultimately govern its progression ^368,369; indeed atherosclerosis rarely occurs in mammals and humans with an LDL-c <80mg/dl ^50,267. Accordingly, in the PESA study of a healthy middle-aged Spanish cohort ³⁰, systemic subclinical atherosclerosis became increasingly rare and undetectable at low LDL-c values, albeit with an increasingly small sample size ³¹. 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 ^60,370,371. Similarly, in humans athero-regression can be induced with intensive lifestyle changes ³⁷² (with <SFAs ^68,190) and/or lipid-lowering drugs ³⁷³ (with LDL-c <80mg/dl ^374,375); and some extreme cases have been reported ^376,377. In fact when considering other mammals, newborn humans and native populations, these low cholesterol levels may even be physiologically normal ^267,324; 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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