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The Remnant Lipoprotein Hypothesis of Diabetes-Associated Cardiovascular Disease

Originally publishedhttps://doi.org/10.1161/ATVBAHA.122.317163Arteriosclerosis, Thrombosis, and Vascular Biology. 2022;42:819–830

Abstract

Both type 1 and type 2 diabetes are associated with an increased risk of atherosclerotic cardiovascular disease (CVD). Research based on human-first or bedside-to-bench approaches has provided new insights into likely mechanisms behind this increased risk. Although both forms of diabetes are associated with hyperglycemia, it is becoming increasingly clear that altered lipoprotein metabolism also plays a critical role in predicting CVD risk in people with diabetes. This review examines recent findings indicating that increased levels of circulating remnant lipoproteins could be a missing link between diabetes and CVD. Although CVD risk associated with diabetes is clearly multifactorial in nature, these findings suggest that we should increase efforts in evaluating whether remnant lipoproteins or the proteins that govern their metabolism are biomarkers of incident CVD in people living with diabetes and whether reducing remnant lipoproteins will prevent the increased CVD risk associated with diabetes.

Highlights

  • APOC3 (apolipoprotein C3) impairs both lipolysis and hepatic clearance of triglyceride-rich lipoproteins and their remnants.

  • APOC3 predicts incident coronary artery events in people with type 1 diabetes.

  • The remnant lipoprotein hypothesis of diabetes-associated cardiovascular disease posits that a reduced clearance rate of remnant lipoprotein particles contributes to the increased cardiovascular disease risk associated with diabetes, in addition to traditional cardiovascular risk factors such as LDL (low-density lipoprotein).

  • Methods to accurately measure and characterize remnant lipoproteins and their metabolism in patients with and without diabetes and mechanistic animal models are urgently needed.

I am delighted and honored to have been selected to deliver the 2021 George Lyman Duff Memorial Lecture. Dr Lyman Duff made seminal contributions to the early field of atherosclerosis. One of his articles relevant to the topic of diabetes and lipid metabolism was published in 1954.1 At that time, 30 years after the discovery of insulin, it was already known that diabetes increases the risk of coronary atherosclerosis, manifested as angina pectoris and coronary thrombosis.2 It was also known that rabbits made diabetic by administration of the β-cell toxin alloxan paradoxically exhibited smaller lesions of atherosclerosis than did nondiabetic rabbits.3 To investigate mechanism, Dr Duff’s group induced diabetes in female rabbits by alloxan, fed them a chow diet coated with cholesterol, and treated some of these rabbits with insulin, analyzing their lipoprotein profiles and atherosclerosis 76 days later. The results showed that cholesterol-fed rabbits whose diabetes was untreated had much higher serum fatty acids in neutral fats (4-fold increase) and serum lipid phosphorus levels (2-fold increase), as compared with nondiabetic controls and insulin-treated diabetic rabbits. Cholesterol levels were similar between the groups, suggesting that untreated diabetic rabbits had large increases in lipoprotein ratios of triglycerides and phospholipids to cholesterol. However, atherosclerosis curiously tended to be reduced in untreated diabetic rabbits as compared with nondiabetic controls and insulin-treated diabetic rabbits.

We now understand that the rabbit alloxan diabetes model is associated with generation of very large TRL (triglyceride-rich lipoprotein) particles that appear to be mostly unable to enter the artery wall, as suggested by Nordestgaard and Zilversmit4 in 1988, although other abnormalities of these large TRLs have not been ruled out. Large TRL particles are believed to be unable to effectively initiate lesions of atherosclerosis.5 The response-to-retention hypothesis of early atherogenesis posited by Williams and Tabas6 in 1995 states that retention of LDL (low-density lipoprotein; a lipoprotein particle much smaller in size than TRLs) in the artery wall is a key early event in lesion initiation.7 This hypothesis was based on earlier work by many investigators8–14 and has been validated by a large number of subsequent studies.

A parallel line of research supported the hypothesis that remnant lipoprotein particles (RLPs) derived from TRLs also contribute to atherosclerosis initiation.15 RLPs are smaller in size than their parent TRLs (chylomicrons and VLDL [very-large-density lipoprotein]) and are believed to be highly atherogenic.16 Accordingly, people with the rare condition dysbetalipoproteinemia (also known as remnant removal disease or type III hyperlipoproteinemia) who are also hyperlipidemic have an increased risk of cardiovascular disease (CVD).17 This condition is often associated with an APOE2/APOE2 genotype. The APOE2 isoform has a low affinity for receptors needed for hepatic uptake of RLPs. Thus, when subjects with the APOE2/APOE2 genotype become hyperlipidemic because of another primary or secondary abnormality of lipid metabolism, RLPs accumulate. There is evidence that both LDLs and RLPs are present in human lesions of atherosclerosis.18

The atherogenicity of RLPs has been explained by their greater cholesterol content per particle vis-à-vis LDL19 and the finding that native and oxidized RLPs promote more extensive cholesterol accumulation by macrophages than LDL.20,21 Moreover, once RLPs have entered the lesion, LPL (lipoprotein lipase) expression by macrophages can lead to generation of yet smaller RLPs22 that are effectively taken up by macrophages. Severely impaired triglyceride lipolysis, as likely explains the predominance of large TRL particles in Dr Duff’s rabbit experiments, would be accompanied by a reduced generation of atherogenic RLPs. Therefore, the early findings by Dr Duff may be consistent with an important role for RLPs in atherogenesis in the setting of diabetes—a topic covered in more detail below.

In this brief review, I will examine recent data leading to the proposal of “the remnant lipoprotein hypothesis of diabetes-associated cardiovascular disease.” I will primarily focus on type 1 diabetes (T1D) and what we have learned about the mechanisms whereby T1D mediates an increased CVD risk by using a human-first (also known as bedside-to-bench or reverse translation) approach. A human-first approach is when human samples, for example from cardiovascular outcome trials involving subjects with T1D‚ are used to identify predictors of CVD risk and subsequently mouse models or cell culture studies are used to reveal whether the identified target is in the causal pathway of diabetes-accelerated atherosclerosis and its mechanisms of action. It is my hope that Lyman Duff would have been interested to know the many new questions and research avenues his research has inspired. It is also my hope that this review will inspire others to pursue studies in this area of research using the sophisticated methodology available in the 21st century.

Hyperglycemia Is Not Sufficient to Explain the Increased CVD Risk Associated With Diabetes

A vast number of publications since the discovery of the link between diabetes and atherosclerosis have centered on hyperglycemia as the culprit mediator of this increased risk. This is logical because hyperglycemia is a consequence of insufficient insulin secretion and impaired insulin action/insulin resistance in T1D and type 2 diabetes (T2D), and both types of diabetes are associated with an increased risk of atherosclerotic CVD.23 Moreover, the DCCT/EDIC study (Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications) on subjects with T1D showed that intensive insulin treatment is associated with a reduced risk of CVD for years after discontinuation of the treatment and that HbA1c (glycated hemoglobin A1) is a strong risk factor for CVD second only to age in T1D24,25 (Figure). Epigenetic phenomena termed metabolic memory or legacy effects have been implicated in the long-term effects of poor glycemic control in human subjects in the DCCT/EDIC study of T1D.26 However, over time, traditional CVD risk factors, such as pulse, LDL cholesterol, triglycerides, and albumin excretion rate, together mediate up to ≈50% of the effect of less tight glycemic control on the risk of CVD in that study.24,27

Figure.

Figure. Using human data on cardiovascular risk associated with type 1 diabetes (T1D) to guide mechanistic studies in mouse models. Human studies have shown that age and HbA1c (glycated hemoglobin A1; a marker of suboptimal glycemic control) are the strongest risk factors for incident cardiovascular disease (CVD) in people with T1D. Other risk factors that associate significantly with CVD risk are (in alphabetic order) APOC3 (apolipoprotein C3), blood pressure, low HDL (high-density lipoprotein) cholesterol and altered HDL composition, LDL (low-density lipoprotein) cholesterol, renal disease, smoking, triglycerides, and white blood cell (WBC) count. Research in mouse models of T1D-associated lesion initiation and progression has demonstrated that APOC3 is in the causal pathway of diabetes-accelerated atherosclerosis, likely working by slowing the clearance of atherogenic remnant lipoprotein particles (RLPs) derived from TRLs (triglyceride-rich lipoproteins). The effect of diabetes on plasma APOC3 levels is due to insulin deficiency or reduced hepatic insulin action rather than due to hyperglycemia. In mouse models in which diabetes is associated with myelopoiesis, hyperglycemia contributes to the increased levels of circulating myeloid cells (monocytes and neutrophils), leading to increased lesion progression and reduced lesion regression through a pathway that involves the RAGE (receptor for advanced glycation end products) in bone marrow progenitor cells and release of the damage-associated molecular pattern proteins S100A8 and S100A9 from neutrophils. However, increased myelopoiesis is not required for diabetes to exacerbate atherosclerosis, and hyperglycemia is not sufficient to explain the increased CVD risk associated with diabetes. Diabetes also results in cholesteryl ester accumulation in macrophages through an APOC3-dependent pathway and causes dampened macrophage metabolism—macrophage phenotypes likely to contribute to worsened lesion morphology, including necrotic core expansion. CVD risk factors in people with T1D that play causal roles in mouse models of T1D-associated atherosclerosis are highlighted by red font. More research is needed to establish whether similar mechanisms apply to type 2 diabetes. Created with BioRender.com.

These observations in humans raise the important question: does hyperglycemia have direct proatherogenic effects or is the association between improved glycemic control and prevention of CVD events largely mediated by covariates affected by suboptimally controlled diabetes? This question has been addressed by several different approaches in mechanistic animal models. The early rabbit experiments performed by Dr Duff already indicated that hyperglycemia is an insufficient explanation for increased atherosclerosis because the untreated diabetic rabbits, who were presumably severely hyperglycemic, failed to exhibit increased atherosclerosis as compared with the nondiabetic control rabbits.1 Another approach to investigate the role of hyperglycemia is the use of SGLT2 (sodium glucose cotransporter 2) inhibition. SGLT2 inhibitors, which are now in clinical use for glucose lowering in patients with T2D and are sometimes used off-label in patients with T1D, lower blood glucose levels by increasing glucose excretion via the urine. SGLT2 inhibition has convincingly been shown to prevent the detrimental effects of diabetes on regression of lesions of atherosclerosis in mouse models28 and to prevent atherosclerosis progression in diabetic mice,29 effects that were attributed to the ability of SGLT2 inhibition to prevent myelopoiesis associated with diabetes in those models (Figure). A few issues complicate data interpretation, however. First, clinical data suggest that the main beneficial effect of SGLT2 inhibitors in humans is to prevent heart failure rather than atherosclerotic disease and that SGLT2 inhibitors are effective in preventing cardiovascular events also in subjects without diabetes30–32 through an effect that is likely not explained by glucose lowering.33 In addition, inhibition of SGLT2 lowers triglycerides and increases LDL levels through a mechanism that involves increased LPL activity through reduced expression of the LPL inhibitor ANGPTL4 (angiopoietin-like 4).34 In the study in which SGLT2 inhibition prevented atherosclerosis progression,29 the atheroprotective effect was suggested to be due, in part, to increased lipoprotein clearance in diabetic Ldlr−/− (LDLR [LDL receptor]-deficient) mice. The beneficial effects of SGLT2 inhibitors on CVD in humans and atherosclerosis in mouse models are, therefore, not necessarily supportive of a major role of elevated glucose in diabetic vascular disease. Another approach that has been used to investigate the role of cellular glucose uptake is to delete the glucose transporter GLUT1 (glucose transporter 1) in specific cell types involved in atherogenesis. Although this approach has generated interesting data demonstrating a causative role for myeloid cell GLUT1 expression in atherosclerosis in a mouse model of transient intermittent hyperglycemia,35 reducing glucose uptake does not necessarily address the effect of diabetes.

Dogma predicts that hyperglycemia increases glucose uptake in cells unable to downregulate glucose transporters in response to elevated levels of extracellular glucose, such as endothelial cells.36,37 The increased glucose uptake can lead to generation of mitochondrial reactive oxygen species and subsequent proatherogenic changes in such cells.36 Conversely, other cells involved in atherogenesis—smooth muscle cells37 and macrophages38—exhibit reduced GLUT1 protein expression and reduced glucose uptake when exposed to elevated glucose levels. Moreover, these cells appear to be largely insensitive to elevated glucose uptake even when forced to take in more glucose through overexpression of GLUT1. Our studies have shown that increased GLUT1 expression in myeloid cells or smooth muscle cells is insufficient for achieving exacerbated atherosclerosis in nondiabetic Ldlr−/− mice.39,40 Similar studies of forced GLUT1 overexpression in endothelial cells would be interesting but have not yet been reported.

Importantly, myeloid progenitor cells appear to be sensitive to hyperglycemia.28,35,41 Several studies have shown that in mouse models in which diabetes leads to increased myelopoiesis, hyperglycemia or transient intermittent hyperglycemia is associated with increased atherosclerosis or impaired lesion regression—an effect likely mediated by the increased levels of circulating monocytes and neutrophils,28,35,42 as shown in the Figure. Another recent mouse study supports the effect of poor glycemic control on long-term detrimental effects on atherosclerosis through a process termed trained immunity.41 It was suggested that elevated glucose leads to epigenetic changes in bone marrow progenitor cells, which translate to increased atherosclerosis when these cells are injected into nondiabetic mice. It is noteworthy, however, that diabetes accelerates atherosclerosis even in the absence of increased myelopoiesis,43 the mechanisms of which appear to be due to insulin deficiency and changes in lipoprotein metabolism as shown in the Figure and discussed below.

Increased formation of AGEs (advanced glycation end products) in response to increased oxidative pressure and hyperglycemia in diabetes has also been extensively investigated in relation to diabetes complications. AGEs can mediate cellular effects through activating RAGE (receptor for AGEs).44 Although RAGE was first identified as a receptor for AGEs, it was later recognized to bind several other ligands, including members of the S100 family (S100A12 and S100B), high mobility group box protein 1, lysophosphatidic acid, and phosphatidylserine. It has, therefore, become apparent that RAGE plays a wider role in inflammatory states and that its actions are not limited to diabetes or hyperglycemia, although it does play an important role in diabetic atherosclerosis in mice.44–46 Another possibility is that AGE-mediated stiffening of arteries contributes to CVD risk together with traditional risk factors because skin intrinsic fluorescence, a reflection of AGEs, associates with CVD in a small study of subjects with long-duration T1D.47 Moreover, a recent study suggested that hyperglycemia activates the mechanosensory ion channel Piezo1 in blood cells and hematopoietic stem cells, leading to thrombosis in T2D.48

Finally, as discussed below, normalization of the impaired TRL clearance associated with diabetes is sufficient for the prevention of atherosclerosis in severely hyperglycemic mouse models,43,49,50 demonstrating that lipid abnormalities override any proatherogenic effects of hyperglycemia. Perhaps hyperglycemia exacerbates the effects of other CVD risk factors? Overall, clinical data and results on mouse models cast doubt on hyperglycemia as a sufficient explanation for the association of diabetes with increased CVD risk.51–53

Inflammation and Atherosclerosis Associated With Diabetes: Evidence Gathering Phase

Myeloid cells, such as monocytes and macrophages, play important roles in all stages of atherosclerosis in humans and animal models.54 Neutrophils have also received increased interest in relation to atherosclerosis in diabetes as these cells appear to respond to hyperglycemia.35,55 Human studies have shown that circulating peripheral blood mononuclear cells and monocytes from subjects recently diagnosed with T1D (<1 year from diabetes onset) had a higher number of cells spontaneously expressing and secreting the cytokines IL (interleukin)-1β and IL-6, as compared with healthy controls and subjects with T2D.56 It is possible that the inflammatory activation of myeloid cells is most pronounced in patients with T1D early after onset of diabetes, associated with the autoimmune response. This possibility might explain the lack of increased expression of IL-6 and IL-1β in other studies performed in subjects later after T1D onset.57 A more recent study investigated differential expression of microRNAs in monocytes from >1000 subjects with T2D, as compared with controls.58 Interestingly, the predicted genes targeted by micoRNAs enriched in T2D belonged to key nodes of cholesterol metabolism rather than to inflammation, suggesting a more pronounced response of monocytes to differences in lipids than inflammation, at least in subjects with T2D.

The comparison of individuals with and without diabetes is less relevant than the comparison between people with diabetes who later will have a CVD event and people with diabetes who have not had a CVD event during a specified follow-up period. Few such analyses have been performed in relation to inflammatory mediators. In one cross-sectional study, blood neutrophil numbers, which associated with plasma S100A9 levels, were significantly elevated in subjects with T1D who had experienced a coronary artery disease event as compared with those who had not.28 Prospective studies on the role of inflammation in incident CVD in subjects with T1D or T2D are needed. In one such prospective study on humans with T1D, blood leukocyte count was a predictor of a first major CVD event, defined as CVD death, myocardial infarction, and stroke, indicating a potential causal role of inflammation.59 However, this association was suggested to be due to the higher rate of smoking in subjects who later had a major CVD event.59 This study did not investigate association of specific leukocyte populations with CVD risk.

A few cardiovascular outcome trials have investigated the causative role of inflammation in incident CVD, and some of these trials included a sufficient number of subjects with diabetes (T2D). CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcome Study) provided strong causal evidence that inflammation contributes to CVD risk in humans with well-controlled levels of LDL cholesterol.60 Subjects with a previous myocardial infarction and elevated levels of high-sensitivity C-reactive protein (≥2 mg/L) were treated with an IL-1β blocking antibody and were followed for a median period of 3.7 years. The IL-1β blockade significantly reduced the recurrent rate of major CVD events (cardiovascular death, myocardial infarction, or stroke) but also increased the incidence of fatal infection.60 Subjects with diabetes had a similar reduction in CVD events as those without diabetes,61 suggesting that the IL-1β pathway may not be of relative greater importance in high-risk subjects with T2D. Moreover, methotrexate (an anti-inflammatory agent used to treat arthritis) did not reduce plasma IL-1β levels and did not protect against second major CVD events in the CIRT (Cardiovascular Inflammation Reduction Trial), which included subjects with T2D or metabolic syndrome.62 It is, therefore, possible that the inflammasome–IL-1β pathway is of particular importance in mediating CVD risk or that the differing results were due to differences in study populations. COLCOT (Colchicine Cardiovascular Outcomes Trial), another positive study using an anti-inflammatory agent (colchicine) in a high-risk population with a recent myocardial infarction, included only 20% of subjects with diabetes, and no subgroup analysis on subjects with diabetes was reported.63

Previous work in our laboratory and those of others have concluded that macrophages in diabetic mice exhibit a more inflammatory phenotype, as compared with cells in nondiabetic controls.28,64–67 Some of these studies used mature thioglycollate-elicited peritoneal macrophages purified by adhesion to tissue culture plates to conclude that macrophages from diabetic mice express and release increased levels of cytokines.64,67 However, low contamination of the adherent macrophages with other cell types might have confounded data interpretation. Recent work in our group on thioglycollate-elicited peritoneal macrophages first sorted by magnetic beads and then adhesion purified showed a higher macrophage purity and a reduced expression of Cd19 mRNA, a B-cell marker, as compared with cells isolated by adhesion purification alone.68

Given these findings, do mature macrophages from diabetic mice take on a more inflammatory phenotype? We recently addressed this question by performing a global proteomics screen on thioglycollate-elicited peritoneal macrophages isolated and purified by the more stringent method noted above.38 Although this method does not rule out differential contamination by other cell types, it mitigates this issue; the cultures were 85% to 90% pure as assessed by flow cytometry of the macrophage markers CD11B (cluster of differentiation molecule 11B) and F4/80.38 These studies showed enrichment of proteins in pathways linked to inflammation in macrophages freshly isolated from diabetic mice, as compared with nondiabetic mice, but the enrichment was related to interferon signaling rather than to proteins involved in the prototypical NF-κB (nuclear factor kappa B) pathway of inflammation.38 Moreover, although proteins involved in interferon signaling pathways were enriched in macrophages from diabetic mice, these cells showed a blunted response to exogenous interferon-β. One weakness of this study is that mature thioglycollate-elicited peritoneal macrophages might not behave as macrophages in lesions of atherosclerosis. Therefore, the effect of diabetes on lesion macrophages (defined as CD68+ cells) has been investigated by laser capture microdissection and subsequent gene expression analyses. Such studies have shown increased gene expression of inflammatory mediators and their receptors in CD68+ cells from models of atherosclerosis regression in diabetic mice.28,66 In these models, diabetes is associated with monocytosis and increased monocyte recruitment into the lesions of atherosclerosis, as compared with regressing lesions in nondiabetic mice. Future single-cell RNA-sequencing studies on lesion cells should provide a better understanding of the inflammatory signatures in different immune cell populations in response to diabetes.

The finding that Il1b mRNA levels are increased in lesion macrophages in diabetic mice28,66 prompted an interest in the role of inflammasomes in diabetes-associated atherosclerosis. The NLRP3 (nod-like receptor family pyrin domain containing 3) inflammasome leads to activation of caspase 1, which in turn converts pro-IL-1β and pro-IL-18 to mature forms that are released from cells. A recent study tested the role of NLRP3 in diabetes-accelerated atherosclerosis in Apoe−/− (apolipoprotein E–deficient) mice rendered diabetic by the β-cell toxin streptozotocin by using the small-molecule NLRP3 inhibitor MCC950.69 Diabetic mice had elevated cholesterol levels concomitant with increased aortic atherosclerosis and increased aortic sinus necrotic cores, as compared with nondiabetic mice, but diabetic mice treated with MCC950 were largely protected from the increased atherosclerosis progression, indicating a role for the NLRP3 inflammasome pathway in diabetes-accelerated atherosclerosis, at least in that mouse model.

Overall, myeloid cells play critical roles in diabetes-accelerated atherosclerosis and diabetes-suppressed lesion regression. Even though myelopoiesis (when present) contributes to increased recruitment of monocytes into lesions, diabetes promotes both lesion initiation and lesion progression, characterized by increased necrotic core expansion, in the absence of myelopoiesis.43 What is certain is that more work is needed to establish whether increased inflammation contributes significantly to increased CVD risk in diabetes in humans and if so, which specific inflammatory pathways are affected.51

Growing Evidence for Beneficial Effects of Insulin on Atherosclerosis

Insulin has critical effects on lipid metabolism, as well as direct effects on cells involved in lesions of atherosclerosis. Thus, T2D is associated with elevated plasma levels of VLDL, lower levels of HDL (high-density lipoprotein) cholesterol, and a shift in the LDL population toward small dense LDL.70,71 Lipoprotein abnormalities are also observed in poorly controlled patients with T1D, primarily manifested as increased plasma triglyceride levels,71 presumably due to increased levels of TRLs and their remnants. These lipid abnormalities are due to a relative lack of insulin or insulin resistance. Importantly, individuals with T1D also develop insulin resistance when overweight or obese, like people without T1D. Moreover, obesity in T1D is associated with dyslipidemia and increased CVD risk.72,73

An important role for insulin in lipoprotein metabolism was highlighted by the generation of a mouse model in which the insulin receptor was deleted specifically in hepatocytes—a target cell type of insulin action that normally expresses high levels of insulin receptors. Strikingly, hepatocyte insulin receptor–deficient mice exhibit increased levels of cholesterol-rich VLDL, IDL (intermediate-density lipoprotein), and LDL, and they develop atherosclerosis even in the presence of LDLRs.74 Thus, deletion of the insulin receptor in hepatocytes is sufficient for the development of dyslipidemia. Furthermore, knockdown of hepatic insulin receptors by short hairpin RNA delivered by an adenovirus in ob/ob mice (a leptin-deficient model of T2D) caused reduced levels of hepatic LDLR, supporting an important role for insulin in maintaining LDLR expression and hepatic APOB (apolipoprotein B)-lipoprotein clearance.75 Conversely, treatment of Apoe−/− mice with exogenous insulin prevented atherosclerosis by reducing cholesterol in the LDL/IDL fractions and increasing LPL activity.76 The ability of insulin to increase LPL activity is mediated, at least in part, by suppression of expression of the LPL inhibitor ANGPTL4.34 Consistent with a role for insulin in maintaining LPL activity, diabetes suppresses tissue LPL activity.50,77

A recent study demonstrated that hepatic insulin receptors also play a role in preventing intestinal cholesterol absorption.78 This effect of hepatic insulin receptors was due to suppression of 12α-hydroxylated bile acid production78 mediated by suppression of the transcription factor FoxO1.79 Thus, silencing of the enzyme Cyp8b1, which generates 12α-hydroxylated bile acid, prevented hypercholesterolemia in mice with hepatic deletion of the insulin receptor and in a mouse model of T1D. These findings might be relevant to people with T1D because the cholesterol absorption inhibitor, ezetimibe, normalized cholesterol absorption and LDL cholesterol in patients with T1D.78 However, the benefit of ezetimibe in reducing CVD events in patients with T1D is yet unknown.

Of particular importance to CVD risk in humans with T1D is the finding that insulin suppresses plasma TRLs and RLPs, in part, by suppressing hepatic expression of APOC3 (apolipoprotein C3) and by lowering plasma APOC3 levels in diabetic mice (see below and Figure).43,80 In support of a role for insulin in regulating APOC3 in human subjects with T1D, APOC3 levels are inversely associated with estimated insulin sensitivity.81 Therefore, APOC3 is another insulin-suppressed target implicated in the increased CVD risk associated with T1D43 (Figure).

The role of insulin action in different cell types directly involved in lesions of atherosclerosis has been investigated in mouse models by deleting the insulin receptor by cell type selective deletion strategies.82 Whereas deletion of insulin receptors in endothelial cells increases atherosclerosis (suggesting a protective effect of insulin in these cells),83 deletion of insulin receptors in hematopoietic cells or myeloid cells produced discrepant effects on atherosclerosis,84,85 and deletion of insulin receptors in smooth muscle cells reduced intimal thickening and smooth muscle proliferation after vascular injury.86

We have recently shown that in mature macrophages, insulin deficiency in severely diabetic mice results in a dampening of macrophage metabolism, characterized by reduced glucose uptake, reduced glycolysis, and reduced oxygen consumption.38 Although the mechanism needs further investigation, it was not due to downregulation of GLUT1 or hyperglycemia.38

Together, these studies show that direct effects of insulin on cells involved in atherosclerosis are cell type specific and suggest that the overarching effects of insulin on the liver, via improved lipoprotein metabolism and reduced intestinal cholesterol uptake, override the proatherogenic effects in other cell types.

Increased Remnant Lipoproteins: a Missing Link Between Diabetes and CVD Risk?

LDL cholesterol predicts the first major CVD events in people with T1D, despite the fact that LDL cholesterol levels overall are not elevated in people with T1D versus those without diabetes (Figure).59,71 Therefore, LDL cholesterol lowering consistently reduces atherosclerosis and incident CVD in people with diabetes, but residual risk remains.51 Animal models of diabetes-accelerated atherosclerosis also require sufficient levels of LDL.

The factors and mechanism underlying the residual risk in diabetes have been the focus of much speculation. One possibility is that LDL cholesterol lowering is insufficient in a subset of patients. Another possibility is that nonlipid risk factors that predict major CVD events in people with T1D (suboptimal glucose control, blood pressure, renal disease, and smoking) explain the residual risk (Figure).25,59 Other interesting possibilities that have been considered are increased inflammation (see above), increased triglycerides and RLPs,87 and reduced HDL function.88

Several lines of evidence support the hypothesis that increased levels of RLPs might be a missing link between diabetes and increased CVD risk. First, whereas limited human data support a role for triglyceride reduction in CVD prevention in patients with diabetes, population studies and genetic studies strongly support a relationship between genes that regulate TRL lipolysis and CVD risk.5,51 This stands to reason because nascent TRLs are large (they carry large loads of triglycerides) and, therefore, contribute significantly to plasma triglyceride levels, whereas the RLPs are lipolysis products of TRLs in which triglycerides have been progressively hydrolyzed, giving rise to a range of smaller particles relatively deprived of triglycerides.16 As mentioned above, one of the genes that regulates TRL lipolysis and RLP clearance is APOC3, and loss-of-function or missense mutations in APOC3 are indeed associated with a reduced CVD risk.5,16,89–91 APOC3 prevents the clearance of TRLs and RLPs by inhibiting LPL, leading to slower, less efficient, triglyceride hydrolysis and, therefore, slower generation of smaller RLPs and, in the case of VLDL, conversion to LDL, and by preventing uptake of RLPs via the hepatic receptors LDLR and LDLR-related protein 1.92–94

A reduced ability to remove RLPs through lipolysis and hepatic clearance appears to be particularly important in subjects with T1D susceptible to CVD because serum APOC3 levels have now been shown to predict CVD events in subjects with T1D in 3 separate cohorts when adjusted for a number of traditional CVD risk factors, even though plasma triglyceride levels are often in the normal range in T1D.43,95,96 We showed that serum APOC3 levels strongly predict coronary artery disease events in subjects with T1D in the prospective CACTI study (Coronary Artery Calcification in Type 1 Diabetes) independent of traditional CVD risk factors,43 and similar findings were reported almost simultaneously in subjects with T1D in DCCT/EDIC.95 In the Finnish Diabetic Nephropathy Study, serum APOC3 again predicted major cardiovascular events in subjects with T1D, and the association was independent of sex, diabetes duration, systolic blood pressure, HbA1c, smoking status, LDL cholesterol, use of lipid-lowering medication, and baseline diabetic kidney disease category (or estimated glomerular filtration rate) but was not independent of RLP levels, suggesting that APOC3 worsens CVD risk through increasing RLPs in T1D.96 Interestingly, in subgroup analyses, the association of serum APOC3 with major cardiovascular events was only significant in individuals with albuminuria,96 consistent with kidney disease as a risk factor for CVD events (Figure). The fact that APOC3 is suppressed by insulin43,80 further strengthens the case for its importance in diabetes.

Second, our data demonstrate that silencing of APOC3 expression in an Ldlr−/− mouse model of T1D97 completely prevents the increased atherosclerosis in diabetic mice without improving blood glucose levels.43 Thus, APOC3 acts in the causal pathway of diabetes-accelerated atherosclerosis. The beneficial effect of APOC3 silencing was evident both in early lesions and in advanced lesions, in which APOC3 suppression prevented the necrotic core expansion in diabetic mice. APOC3 silencing did not significantly affect plasma total APOB levels (APOB is a component of TRLs, RLPs, and LDLs). Because most APOB in plasma is carried by LDL, these findings suggest that APOC3 silencing lowers TRLs and their atherogenic remnants in Ldlr−/− mice, consistent with human studies.98 In the same T1D mouse model, APOC3 accumulates in the artery wall in close proximity to APOB and macrophages, suggesting increased arterial accumulation of APOC3-carrying lipoprotein particles, perhaps RLPs, in diabetic mice.43,99 Indeed, APOC3 silencing prevented the increased cholesteryl ester accumulation in macrophages from diabetic mice.43 Conversely, Ldlr−/− mice carrying a human APOC3 transgene have increased atherosclerosis.100 In the mouse model of T1D-accelerated atherosclerosis,97 the increased atherosclerosis is likely due to increased levels of RLPs in circulation, leading to increased trapping of these particles in the artery wall.50 Thus, in addition to APOC3 silencing, forced hepatic expression of the VLDL receptor, which mediates uptake of APOE-containing lipoproteins such as RLPs, or forced hepatic expression of the transcription factor CREBH (cAMP responsive element binding protein 3 like 3), which promotes RLP clearance, in diabetic mice prevented the increased atherosclerosis progression despite persistent hyperglycemia.49,50 In human subjects with T1D, IDL cholesterol was shown to be a better predictor of mortality (the majority of which is due to CVD) than LDL cholesterol.101

Together, these findings support the proposal that RLPs contribute to CVD risk in people with T1D. Whether this is indeed the case would need to be tested using new methods of accurately measuring RLP concentrations and sizes in large clinical studies. Currently, there is no generally agreed upon method of measuring RLPs.16 Clinical studies have used the formula RLP cholesterol=total cholesterol−LDL cholesterol−HDL cholesterol. In such studies, LDL cholesterol is often calculated by the Friedewald formula102 (LDL cholesterol=total cholesterol−HDL cholesterol−triglycerides/5), where the factor triglycerides/5 assumes that virtually all of the plasma triglycerides are carried on VLDL and that the ratio of triglycerides to cholesterol in VLDL is constant at about 5:1. This formula is convenient for large clinical trials but has several limitations. First, it is only valid in fasting samples (in which chylomicrons are absent) and for triglyceride levels between 100 and 400 mg/dL. Moreover, if the Friedewald LDL cholesterol calculation is plugged into the RLP cholesterol formula above, RLP cholesterol is in fact equal to fasting triglycerides/5, the original estimation of VLDL cholesterol.103 Other methods of measuring RLP particle number and size include nuclear magnetic resonance and differential ion mobility analysis.50 Nuclear magnetic resonance is based on computational deconvolution of the proton nuclear magnetic resonance signal of plasma lipid methyl groups characteristic of lipoproteins. Differential ion mobility analysis has the advantage that both the size and concentrations of lipoprotein particles can be accurately determined.104,105 The method is based on the principle that particles of a given size and charge behave in a predictable manner when carried in a laminar flow of air and subjected to an electric field. Differential ion mobility analysis may offer a powerful approach for quantifying RLPs and has recently been used to evaluate RLP levels and sizes in humans.50,106 Analyzing RLPs in mice is even more challenging because there is no clear separation of TRLs, RLPs, and LDL by density ultracentrifugation or differential ion mobility analysis in Ldlr−/− mice. Moreover, contrary to humans who produce APOB48 only in the intestine, making it possible to identify chylomicron remnants by the presence of APOB48 (rather than full-length APOB100-containing VLDL derived from the liver), >75% of serum APOB48 is derived from the liver in mice.107

Overall, the remnant lipoprotein hypothesis of diabetes-associated CVD risk is compelling, but much more research is needed to establish high-throughput clinical methods of quantifying RLP particle concentrations, sizes, and functions and to investigate whether RLPs causally contribute to the increased CVD risk in people living with diabetes.

The Remnant Lipoprotein Hypothesis of Diabetes-Associated CVD: Remaining Questions and Challenges

Three independent studies have confirmed that serum APOC3 predicts CVD events in subjects with T1D.43,95,96 This finding was initially surprising because subjects with T1D often have triglyceride levels in the normal range. Whether this association is also present in subjects with T2D and different levels of triglycerides needs to be investigated. A critical next step is to test whether APOC3 inhibition would prevent CVD risk in patients with T1D or T2D. Several strategies of inhibiting APOC3 have already been developed, including antisense oligonucleotides, small interfering RNAs, and blocking antibodies. A second-generation antisense oligonucleotide therapeutic (volanesorsen) has been shown in clinical trials to lower plasma triglycerides in severely hypertriglyceridemic subjects108–110 and in a small study on subjects with T2D.111 This drug is approved for the treatment of familial chylomicronemia syndrome in the European Union. Since volanesorsen was associated with reduced platelet counts in some subjects,108 a newer liver-targeted N-acetyl galactosamine–conjugated APOC3 antisense oligonucleotide (olezarsen) has recently been tested in clinical trials.112 Promisingly, in a 6- to 12-month clinical trial on subjects with moderate hypertriglyceridemia (200–500 mg/dL) and established CVD, monthly injections of olezarsen reduced plasma triglycerides and VLDL without causing changes in platelet counts.98 Cardiovascular outcome studies using this therapeutic, therefore, appear feasible.

We also need more mechanistic studies on how APOC3 worsens CVD risk, whether this is mediated by RLP accumulation or other mechanisms (such as inflammation113), how diabetes affects RLP metabolism and modification (such as oxidation), structure and function, and the relationship of APOC3 and RLPs to other lipoproteins, such as LDL subpopulations and HDL subpopulations in the setting of diabetes.

In this context, it is of note that a recent phase 2 trial on subjects on statins treated with the new triglyceride-lowering PPARα modulator pemafibrate convincingly showed reduced levels of triglycerides, APOC3, and RLPs.106 Although this 12-week trial suggested that pemafibrate might be effective in preventing CVD events, discontinuation of the large PROMINENT trial (Pemafibrate to Reduce Cardiovascular Outcomes by Reducing Triglycerides in Patients With Diabetes) was announced in April, 2022, because the primary end point (major CVD event) was unlikely to be met. This trial included subjects with T2D and dyslipidemia. How can the failure of this much anticipated trial be understood if RLPs are indeed important in mediating increased residual CVD risk in diabetes? One possible explanation might lie in the fact that APOC3 inhibits the action of LPL, which results not only in reduced lipolysis of TRLs but also in reduced conversion of VLDL-derived RLPs to LDL.92 Accordingly, pemafibrate reduced APOC3 but increased LDL cholesterol (leaving total APOB100 levels unchanged) in the phase 2 trial.106 RLP lowering is unlikely to be effective in preventing CVD risk if it only results in conversion to LDL without efficient removal. RLP lowering leading to increases in LDL cholesterol levels and unchanged total APOB100 levels many not be effective. Release of the final data from PROMINENT is needed before we can better understand the outcome of this trial.

Another interesting question is how impaired RLP clearance in diabetes is associated with changes in HDL structure and function. It is known that subjects with LPL deficiency are unable to efficiently convert TRLs to RLPs and that they also exhibit very low HDL particle concentrations.114 We have recently shown that small HDL particles are dysfunctional (measured by a reduced cholesterol efflux capacity) in subjects with T2D115 and that the protein composition of HDL predicts incident CVD in subjects with T1D.116 Intriguingly, in the latter study, levels of pulmonary surfactant protein (pulmonary surfactant protein B) in HDL predicted incident CVD in a fully adjusted model that controlled for lipids and other risk factors.

Finally, equitable access to health care and diabetes management technology would reduce CVD risk associated with diabetes. Questions related to how to achieve equitable health care in the United States and worldwide for patients with diabetes are of utmost importance. Addressing the challenge of equitable health care access would unquestionably make the biggest impact on reducing CVD death associated with diabetes.

Article Information

Acknowledgments

I am grateful to all my trainees, collaborators, and colleagues who have contributed to the studies on cardiovascular disease risk associated with diabetes in humans and the mechanistic mouse models.

Nonstandard Abbreviations and Acronyms

AGE

advanced glycation end product

ANGPTL4

angiopoietin-like 4

APOB

apolipoprotein B

APOC3

apolipoprotein C3

Apoe−/−

apolipoprotein E deficient

CACTI

Coronary Artery Calcification in Type 1 Diabetes

CANTOS

Canakinumab Anti-Inflammatory Thrombosis Outcome Study

CIRT

Cardiovascular Inflammation Reduction Trial

COLCOT

Colchicine Cardiovascular Outcomes Trial

CVD

cardiovascular disease

DCCT/EDIC

Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications

HDL

high-density lipoprotein

IDL

intermediate-density lipoprotein

IL

interleukin

LDL

low-density lipoprotein

LDLR

low-density lipoprotein receptor

Ldlr−/−

low-density lipoprotein receptor deficient

LPL

lipoprotein lipase

NF-κB

nuclear factor kappa B

NLRP3

nod-like receptor family pyrin domain containing 3

PROMINENT

Pemafibrate to Reduce Cardiovascular Outcomes by Reducing Triglycerides in Patients With Diabetes

RAGE

receptor for advanced glycation end products

RLP

remnant lipoprotein particle

SGLT2

sodium glucose cotransporter 2

T1D

type 1 diabetes

T2D

type 2 diabetes

TRL

triglyceride-rich lipoprotein

VLDL

very-large-density lipoprotein

Disclosures None.

Footnotes

For Sources of Funding and Disclosures, see page 827.

This review article was presented as the “George Lyman Duff Memorial Lecture” in November 2021, virtually, as part of the American Heart Association Scientific Sessions. https://www.ahajournals.org/atvb/lecture-series.

Correspondence to: Karin E. Bornfeldt, PhD, Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, University of Washington Medicine Diabetes Institute, 750 Republican St, University of Washington, Seattle, WA 98109. Email

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