High-Density Lipoprotein in Metabolic Disorders and Beyond: An Exciting New World Full of Challenges and Opportunities.

High-density lipoprotein (HDL) is an enigmatic member of the plasma lipid and lipoprotein transport system, best known for its ability to promote the reverse cholesterol efflux and the unloading of excess cholesterol from peripheral tissues. More recently, data in experimental mice and humans suggest that HDL may play important novel roles in other physiological processes associated with various metabolic disorders. Important parameters in the HDL functions are its apolipoprotein and lipid content, further reinforcing the principle that HDL structure defines its functionality. Thus, based on current evidence, low levels of HDL-cholesterol (HDL-C) or dysfunctional HDL particles contribute to the development of metabolic diseases such as morbid obesity, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. Interestingly, low levels of HDL-C and dysfunctional HDL particles are observed in patients with multiple myeloma and other types of cancer. Therefore, adjusting HDL-C levels within the optimal range and improving HDL particle functionality is expected to benefit such pathological conditions. The failure of previous clinical trials testing various HDL-C-raising pharmaceuticals does not preclude a significant role for HDL in the treatment of atherosclerosis and related metabolic disorders. Those trials were designed on the principle of "the more the better", ignoring the U-shape relationship between HDL-C levels and morbidity and mortality. Thus, many of these pharmaceuticals should be retested in appropriately designed clinical trials. Novel gene-editing-based pharmaceuticals aiming at altering the apolipoprotein composition of HDL are expected to revolutionize the treatment strategies, improving the functionality of dysfunctional HDL.

High density lipoprotein in atherosclerosis and coronary heart disease: Where do we stand today?

Epidemiological studies during the last five years suggest that a relation between high density lipoprotein cholesterol (HDL-C) levels and the risk for cardiovascular disease (CVD) does exist but follows rather a "U-shaped" curve with an optimal range of HDL-C concentration between 40 and 70 mg/dl for men and 50-70 mg/dl for women. Moreover, as research in the field of lipoproteins progresses it becomes increasingly apparent that HDL particles possess different attributes and depending on their structural and functional characteristics, they may be "antiatherogenic" or "proatherogenic". In light of this information, it is highly doubtful that the choice of experimental drugs and the design of respective clinical trials that put the HDL-C raising hypothesis at test, were the most suitable. Here, we compile the existing literature on HDL, providing a critical up-to-date view that focuses on key data from the biochemistry, epidemiology and pharmacology of HDL, including data from clinical trials. We also discuss the most up-to-date information on the contribution of HDL structure and function to the prevention of atherosclerosis. We conclude by summarizing important differences between mouse models and humans, that may explain why pharmacological successes in mice turn out to be failures in humans.

HDL and type 2 diabetes: the chicken or the egg?

HDL is a complex macromolecular cluster of various components, such as apolipoproteins, enzymes and lipids. Quality evidence from clinical and epidemiological studies led to the principle that HDL-cholesterol (HDL-C) levels are inversely correlated with the risk of CHD. Nevertheless, the failure of many cholesteryl ester transfer protein inhibitors to protect against CVD casts doubts on this principle and highlights the fact that HDL functionality, as dictated by its proteome and lipidome, also plays an important role in protecting against metabolic disorders. Recent data indicate that HDL-C levels and HDL particle functionality are correlated with the pathogenesis and prognosis of type 2 diabetes mellitus, a major risk factor for CVD. Hyperglycaemia leads to reduced HDL-C levels and deteriorated HDL functionality, via various alterations in HDL particles' proteome and lipidome. In turn, reduced HDL-C levels and impaired HDL functionality impact the performance of key organs related to glucose homeostasis, such as pancreas and skeletal muscles. Interestingly, different structural alterations in HDL correlate with distinct metabolic abnormalities, as indicated by recent data evaluating the role of apolipoprotein A1 and lecithin-cholesterol acyltransferase deficiency in glucose homeostasis. While it is becoming evident that not all HDL disturbances are causatively associated with the development and progression of type 2 diabetes, a bidirectional correlation between these two conditions exists, leading to a perpetual self-feeding cycle.

Tissue-specific functional interaction between apolipoproteins A1 and E in cold-induced adipose organ mitochondrial energy metabolism.

White (WAT) and brown (BAT) adipose tissue, the two main types of adipose organ, are responsible for lipid storage and non-shivering thermogenesis, respectively. Thermogenesis is a process mediated by mitochondrial uncoupling protein 1 (UCP1) which uncouples oxidative phosphorylation from ATP production, leading to the conversion of free fatty acids to heat. This process can be triggered by exposure to low ambient temperatures, caloric excess, and the immune system. Recently mitochondrial thermogenesis has also been associated with plasma lipoprotein transport system. Specifically, apolipoprotein (APO) E3 is shown to have a bimodal effect on WAT thermogenesis that is highly dependent on its site of expression. Similarly, APOE2 and APOE4 differentially affect BAT and WAT mitochondrial metabolic activity in processes highly modulated by APOA1. Furthermore, the absence of classical APOA1 containing HDL (APOA1-HDL), is associated with no measurable non-shivering thermogenesis in WAT of mice fed high fat diet. Based on these previous observations which indicate important regulatory roles for both APOA1 and APOE in adipose tissue mitochondrial metabolic activity, here we sought to investigate the potential roles of these apolipoproteins in BAT and WAT metabolic activation in mice, following stimulation by cold exposure (7 °C). Our data indicate that APOA1-HDL promotes metabolic activation of BAT only in the presence of very low levels (virtually undetectable) of APOE3-containing HDL (APOE3-HDL), which acts as an inhibitor in this process. In contrast, induction of WAT thermogenesis is subjected to a more complicated regulation which requires the combined presence of both APOA1-HDL and APOE3-HDL.

Τhe Antioxidant Function of HDL in Atherosclerosis.

Atherosclerosis is a multistep process that progresses over a long period of time and displays a broad range of severity. In its final form, it manifests as a lesion of the intimal layer of the arterial wall. There is strong evidence supporting that oxidative stress contributes to coronary heart disease morbidity and mortality and antioxidant high-density lipoprotein (HDL) could have a beneficial role in the prevention and prognosis of the disease. Indeed, certain subspecies of HDL may act as natural antioxidants preventing oxidation of lipids on low-density lipoprotein (LDL) and biological membranes. The antioxidant function may be attributed to inhibition of synthesis or neutralization of free radicals and reactive oxygen species by HDL lipids and associated enzymes or transfer of oxidation prone lipids from LDL and biological membranes to HDL for catabolism. A limited number of clinical trials suggest that the increased antioxidant potential of HDL correlates with decreased risk for atherosclerosis. Some nutritional interventions to increase HDL antioxidant activity have been proposed with limited success so far. The limitations in measuring and understanding HDL antioxidant function in vivo are also discussed.

Isoform and tissue dependent impact of apolipoprotein E on adipose tissue metabolic activation: The role of apolipoprotein A1.

Adipose organ is made of white (WAT) and brown (BAT) adipose tissue which are primarily responsible for lipid storage and energy production (heat and ATP) respectively. Metabolic activation of WAT may ascribe to this tissue characteristics of BAT, namely non-shivering thermogenesis and ATP production. Recent data indicate that apolipoproteins E (APOE) and A1 (APOA1) regulate WAT mitochondrial metabolic activation. Here, we investigated the functional cross-talk between natural human APOE2 and APOE4 isoforms with APOA1 in this process, using Apoe2knock-in and Apoe4knock-in mice. At baseline when Apoe2knock-in and Apoe4knock-in mice express both APOE and Apoa1, the Apoe2knock-in strain appears to have higher mitochondrial oxidative phosphorylation levels and non-shivering thermogenesis in WAT compared to Apoe4knock-in mice. When mice were switched to a high-fat diet for 18 weeks, circulating levels of endogenous Apoa1 in Apoe2knock-in mice became barely detectable though significant levels of APOE2 were still present. This change was accompanied by a significant reduction in WAT mitochondrial Ucp1 expression while BAT Ucp1 was unaffected. Ectopic APOA1 expression in Apoe2knock-in animals potently stimulated WAT but not BAT mitochondrial Ucp1 expression providing further evidence that APOA1 potently stimulates WAT non-shivering thermogenesis in the presence of APOE2. Ectopic expression of APOA1 in Apoe4knock-in mice stimulated BAT but no WAT mitochondrial Ucp1 levels, suggesting that in the presence of APOE4, APOA1 is a trigger of BAT non-shivering thermogenesis. Overall, our data identified a tissue-specific role of the natural human APOE2 and APOE4 isoforms in WAT- and BAT-metabolic activation respectively, that appears dependent on circulating APOA1 levels.

Pleiotropic effects of apolipoprotein A-â ¡ on high-density lipoprotein functionality, adipose tissue metabolic activity and plasma glucose homeostasis.

Apolipoprotein A-Ⅱ (APOA-Ⅱ) is the second most abundant apolipoprotein of high-density lipoprotein (HDL) synthesized mainly by the liver and to a much lesser extent by the intestine. Transgenic mice overexpressing human APOA-Ⅱ present abnormal lipoprotein composition and are prone to atherosclerosis, though in humans the role for APOA-Ⅱ in coronary heart disease remains controversial. Here, we investigated the effects of overexpressed APOA-Ⅱ on HDL structure and function, adipose tissue metabolic activity, glucose tolerance and insulin sensitivity. C57BL/6 mice were infected with an adenovirus expressing human APOA-Ⅱ or a control adenovirus AdGFP, and five days post-infection blood and tissue samples were isolated. APOA-Ⅱ expression resulted in distinct changes in HDL apoproteome that correlated with increased antioxidant and anti-inflammatory activities. No effects on cholesterol efflux from RAW 264.7 macrophages were observed. Molecular analyses in white adipose tissue (WAT) indicated a stimulation of oxidative phosphorylation coupled with respiration for ATP production in mice overexpressing APOA-Ⅱ. Finally, overexpressed APOA-Ⅱ improved glucose tolerance of mice but had no effect on the response to exogenously administered insulin. In summary, expression of APOA-Ⅱ in C57BL/6 mice results in pleiotropic effects with respect to HDL functionality, adipose tissue metabolism and glucose utilization, many of which are beneficial to health.

Adrenoceptor-related decrease in serum triglycerides is independent of PPARα activation.

Adrenoceptor (AR)-linked pathways belong to the major components of the stress response system and are associated with the pathophysiology of diseases within the spectrum of metabolic syndrome. In this study, the role of adrenoceptor stimulation in serum triglyceride (TG) regulation in mice was investigated. For this purpose, α1 -ARs were activated with phenylephrine (PH) and ß1/2 -ARs with isoprenaline (ISOP). Both AR agonists markedly reduced serum TG levels independently of PPARα activation. These drugs also significantly activated the hormone-sensitive lipase in the white adipose tissue indicating increased mobilization of TGs in this tissue. In addition, PH and ISOP up-regulated Lpl, Nr4A, Dgat1, Mttp, Aadac and Cd36 genes, critical in TG regulation, whereas the observed decrease in serum TG levels was independent of the hepatic very low-density lipoprotein (VLDL)-TG secretion. Interestingly, PH and ISOP also inactivated the hepatic insulin/PI3k/AKT/FoxO1 signaling pathway, holding a critical role in the regulation of genes involved in TG synthesis. Taken together, the findings of the present study indicate that stimulation of α1 - and ß1/2 -ARs markedly reduced serum TG steady-state levels as a result of alterations in TG synthesis, uptake, transport, hydrolysis, metabolism and clearance, an effect induced by PPARα independent mechanisms.

Impact of apolipoprotein A1- or lecithin:cholesterol acyltransferase-deficiency on white adipose tissue metabolic activity and glucose homeostasis in mice.

High density lipoprotein (HDL) has attracted the attention of biomedical community due to its well-documented role in atheroprotection. HDL has also been recently implicated in the regulation of islets of Langerhans secretory function and in the etiology of peripheral insulin sensitivity. Indeed, data from numerous studies strongly indicate that the functions of pancreatic ß-cells, skeletal muscles and adipose tissue could benefit from improved HDL functionality. To better understand how changes in HDL structure may affect diet-induced obesity and type 2 diabetes we aimed at investigating the impact of Apoa1 or Lcat deficiency, two key proteins of peripheral HDL metabolic pathway, on these pathological conditions in mouse models. We report that universal deletion of apoa1 or lcat expression in mice fed western-type diet results in increased sensitivity to body-weight gain compared to control C57BL/6 group. These changes in mouse genome correlate with discrete effects on white adipose tissue (WAT) metabolic activation and plasma glucose homeostasis. Apoa1-deficiency results in reduced WAT mitochondrial non-shivering thermogenesis. Lcat-deficiency causes a concerted reduction in both WAT oxidative phosphorylation and non-shivering thermogenesis, rendering lcat-/- mice the most sensitive to weight gain out of the three strains tested, followed by apoa1-/- mice. Nevertheless, only apoa1-/- mice show disturbed plasma glucose homeostasis due to dysfunctional glucose-stimulated insulin secretion in pancreatic ß-islets and insulin resistant skeletal muscles. Our analyses show that both apoa1-/- and lcat-/- mice fed high-fat diet have no measurable Apoa1 levels in their plasma, suggesting no direct involvement of Apoa1 in the observed phenotypic differences among groups.

Pharmacological Management of Dyslipidemia in Atherosclerosis: Limitations, Challenges, and New Therapeutic Opportunities.

Clinical and epidemiological studies during the last 7 decades indicated that elevated low-density lipoprotein cholesterol (LDL-C) levels and reduced high-density lipoprotein cholesterol (HDL-C) levels correlate with the pathogenesis and progression of atherosclerotic lesions in the arterial wall. This observation led to the development of LDL-C-lowering drugs for the prevention and treatment of atherosclerosis, some with greater success than others. However, a body of recent clinical evidence shows that a substantial residual cardiovascular risk exists even at very low levels of LDL-C, suggesting that new therapeutic modalities are still needed for reduction of atherosclerosis morbidity and mortality. Unfortunately, HDL-C-raising drugs developed toward this goal had disappointing results thus far. Here, we critically review the literature presenting available evidence and challenges that need to be met and discuss possible new avenues for the development of novel lipid pharmacotherapeutics to reduce the burden of atherosclerosis.

The olive constituent oleuropein, as a PPARα agonist, markedly reduces serum triglycerides.

Oleuropein (OLE), a main constituent of olive, exhibits antioxidant and hypolipidemic effects, while it reduces the infarct size in chow- and cholesterol-fed rabbits. Peroxisome proliferator-activated receptor α (PPARα) has essential roles in the control of lipid metabolism and energy homeostasis. This study focused on the mechanisms underlying the hypolipidemic activity of OLE and, specifically, on the role of PPARα activation in the OLE-induced effect. Theoretical approach using Molecular Docking Simulations and luciferase reporter gene assay indicated that OLE is a ligand of PPARα. The effect of OLE (100 mg/kg, p.o., per day, ×6 weeks) on serum triglyceride (TG) and cholesterol levels was also assessed in adult male wild-type and Ppara-null mice. Molecular Docking Simulations, Luciferase reporter gene assay and gene expression analysis indicated that OLE is a PPARα agonist that up-regulates several PPARα target genes in the liver. This effect was associated with a significant reduction of serum TG and cholesterol levels. In contrast, OLE had no effect in Ppara-null mice, indicating a direct involvement of PPARα in the OLE-induced serum TG and cholesterol reduction. Activation of hormone-sensitive lipase in the white adipose tissue (WAT) and the liver of wild-type mice and up-regulation of several hepatic factors involved in TG uptake, transport, metabolism and clearance may also contribute in the OLE-induced TG reduction. In summary, OLE has a beneficial effect on TG homeostasis via PPARα activation. OLE also activates the hormone sensitive lipase in the WAT and liver and up-regulates several hepatic genes with essential roles in TG homeostasis.

Site-specific effects of apolipoprotein E expression on diet-induced obesity and white adipose tissue metabolic activation.

Apolipoprotein E (APOE) has been strongly implicated in the development of diet induced obesity. In the present study, we investigated the contribution of brain and peripherally expressed human apolipoprotein E3 (APOE3), the most common human isoform, to diet induced obesity. In our studies APOE3 knock-in (Apoe3knock-in), Apoe-deficient (apoe-/-) and brain-specific expressing APOE3 (Apoe3brain) mice were fed western-type diet for 12week and biochemical analyses were performed. Moreover, AAV-mediated gene transfer of APOE3 to apoe-/- mice was employed, as a means to achieve APOE3 expression selectively in periphery, since peripherally expressed APOE does not cross blood brain barrier (BBB) or blood-cerebrospinal fluid barrier (BCSFB). Our data suggest a bimodal role of APOE3 in visceral white adipose tissue (WAT) mitochondrial metabolic activation that is highly dependent on its site of expression and independent of postprandial dietary lipid deposition. Our findings indicate that brain APOE3 expression is associated with a potent inhibition of visceral WAT mitochondrial oxidative phosphorylation, leading to significantly reduced substrate oxidation, increased fat accumulation and obesity. In contrast, peripherally expressed APOE3 is associated with a notable shift of substrate oxidation towards non-shivering thermogenesis in visceral WAT mitochondria, leading to resistance to obesity.

Apolipoprotein E in diet-induced obesity: a paradigm shift from conventional perception.

Apolipoprotein E (APOE) is a major protein component of peripheral and brain lipoprotein transport systems. APOE in peripheral circulation does not cross blood brain barrier or blood cerebrospinal fluid barrier. As a result, peripheral APOE expression does not affect brain APOE levels and vice versa. Numerous epidemiological studies suggest a key role of peripherally expressed APOE in the development and progression of coronary heart disease while brain APOE has been associated with dementia and Alzheimer's disease. More recent studies, mainly in experimental mice, suggested a link between Apoe and morbid obesity. According to the latest findings, expression of human apolipoprotein E3 (APOE3) isoform in the brain of mice is associated with a potent inhibition of visceral white adipose tissue (WAT) mitochondrial oxidative phosphorylation leading to significantly reduced substrate oxidation, increased fat accumulation and obesity. In contrast, hepatically expressed APOE3 is associated with a notable shift of substrate oxidation towards non-shivering thermogenesis in visceral WAT mitochondria, leading to resistance to obesity. These novel findings constitute a major paradigm shift from the widely accepted perception that APOE promotes obesity via receptor-mediated postprandial lipid delivery to WAT. Here, we provide a critical review of the latest facts on the role of APOE in morbid obesity.

Advances in high-density lipoprotein physiology: surprises, overturns, and promises.

Emerging evidence strongly supports that changes in the HDL metabolic pathway, which result in changes in HDL proteome and function, appear to have a causative impact on a number of metabolic disorders. Here, we provide a critical review of the most recent and novel findings correlating HDL properties and functionality with various pathophysiological processes and disease states, such as obesity, type 2 diabetes mellitus, nonalcoholic fatty liver disease, inflammation and sepsis, bone and obstructive pulmonary diseases, and brain disorders.

Deficiency in apolipoprotein A-I ablates the pharmacological effects of metformin on plasma glucose homeostasis and hepatic lipid deposition.

Recently, we showed that deficiency in apolipoprotein A-I (ApoA-I) sensitizes mice to diet-induced obesity, glucose intolerance and NAFLD. Here we investigated the potential involvement of ApoA-I in the pharmacological effects of metformin on glucose intolerance and NAFLD development. Groups of apoa1-deficient (apoa1(-/-)) and C57BL/6 mice fed western-type diet were either treated with a daily dose of 300 mg/kg metformin for 18 weeks or left untreated for the same period. Then, histological and biochemical analyses were performed. Metformin treatment led to a comparable reduction in plasma insulin levels in both C57BL/6 and apoa1(-/-) mice following intraperitoneal glucose tolerance test. However, only metformin-treated C57BL/6 mice maintained sufficient peripheral insulin sensitivity to effectively clear glucose following the challenge, as indicated by a [(3)H]-2-deoxy-D-glucose uptake assay in isolated soleus muscle. Similarly, deficiency in ApoA-I ablated the effect of metformin on hepatic lipid deposition and NAFLD development. Gene expression analysis indicated that the effects of ApoA-I on metformin treatment may be independent of adenosine monophosphate-activated protein kinase (AMPK) activation and de novo lipogenesis. Interestingly, metformin treatment reduced mitochondrial oxidative phosphorylation function only in apoa1(-/-) mice. Our data show that the role of ApoA-I in diabetes extends to the modulation of the pharmacological actions of metformin, a common drug for the treatment of type 2 diabetes.

Scavenger Receptor Class B Type I Regulates Plasma Apolipoprotein E Levels and Dietary Lipid Deposition to the Liver.

Scavenger receptor class B type I (SR-BI) is primarily responsible for the selective uptake of cholesteryl esters (CE) of high-density lipoprotein (HDL) by the liver and other tissues. In the present study, we show that SR-BI-deficient (scarb1(-/-)) mice are resistant to diet-induced obesity, hepatic lipid deposition, and glucose intolerance after 24 weeks of being fed a western-type diet. No differences in energy expenditure or mitochondrial function could account for the observed phenotype. Kinetic and gene expression analyses suggested reduced de novo fatty acid synthesis in scarb1(-/-) mice. Furthermore, adenosine monophosphate-activated protein kinase (AMPK)-stimulated hepatic FFA catabolism was reduced in these mice, leaving direct dietary lipid uptake from plasma as the major modulator of hepatic lipid content. Analysis of the apolipoprotein composition of plasma lipoproteins revealed a significant accumulation of apolipoprotein E (ApoE)-containing HDL and TG-rich lipoproteins in scarb1(-/-) mice that correlated with reduced plasma LpL activity. Our data suggest that scarb1(-/-) mice fed a western-type diet for 24 weeks accumulate CE- and ApoE-rich HDL of abnormal density and size. The elevated HDL-ApoE levels inhibit plasma LpL activity, blocking the clearance of triglyceride-rich lipoproteins and preventing the shuttling of dietary lipids to the liver.

HDL quality and functionality: what can proteins and genes predict?

Epidemiological and clinical studies have over the years established that dyslipidemia constitutes the main risk factor for atherosclerosis. The inverse correlation between HDL cholesterol (HDL-C) levels and coronary heart disease morbidity and mortality identified HDL-C as an alternative pharmacological target to LDL-C and a potential anti-atherosclerosis marker. However, more recent data reinforced the principle of 'HDL quality' in atherosclerosis that refers to the functionality of HDL particle, as defined by its protein and lipid content, rather than HDL-C levels in plasma. Since HDL functionality depends on the genes and proteins of the HDL metabolic pathway, its apoprotein composition may serve as a surrogate marker of atheroprotection. In this manuscript we review the atheroprotective properties of HDL in relation to the proteins of HDL metabolic pathway and discuss what HDL-associated genes and proteins may reveal about HDL functionality in the assessment of coronary risk.