Monday June 27 Plenary session:

Pathophysiology and treatment of dyslipidemias

New insights into the aetiology and pathophysiology of hereditary dyslipidemias

Professor Helen H. Hobbs, Professor of Internal Medicine and Molecular Genetics and Chief of Clinical Genetics at the University of Texas Southwestern Medical Center, Dallas, USA

Recent evidence implicates a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) with the development of a spectrum of diseases ranging from hepatocellular carcinoma, to non-alcoholic fatty liver disease (NAFLD). NAFLD affects about one-third of adults and an increasing number of children in developed countries.

Professor Hobbs presented data on this variant of PNPLA3, from a study in African-Americans, Caucasian Americans and Hispanics. This PNPLA3 variant increased triglycerides and inflammation, leading to hepatic steatosis, but was not associated with body mass index or insulin resistance. The pathogenesis underlying this link is not yet fully understood.

In the Dallas Heart Study, the presence of the PNPLA3 variant was associated with fatty liver disease progression, as evidenced by stepwise increases in alanine transaminase (ALT) levels. Individuals who had two 148M alleles had a 52% increase in ALT levels compared with those with two 148I alleles. Liver steatosis was also more prevalent in these carriers of two 148M alleles. However, glucose tolerance and insulin sensitivity were similar across all three genotypes.

However, PNPLA3 may not be the only genetic variant that contributes to liver disease. A number of genome-wide association studies have found that PNPLA3 and other genomic variations are associated with hepatic steatosis, including another lipase and kinase regulatory proteins. However, while PNPLA3 has a direct effect on hepatic triglyceride content, this effect is modified by the interaction between gene and environment. In related research, the family of one individual in the Dallas Heart Study with fatty liver disease is being studied, to try to understand the phenotype for PNPLA3 and how to prevent and treat fatty liver disease. Professor Hobbs said; ‘By avoiding weight gain it is possible to avoid disease progression in these individuals carrying these variants.’

Evidence from a knockout model suggests that the mutant may be a gain of function mutation.

Understanding the role of GPIHBP1 in lipid metabolism

Professor Stephen Young, Professor of Medicine and Director, Cellular and Molecular Cardiology Unit, David Geffen School of Medicine at UCLA, USA

Lipoprotein lipase (LPL) is a key enzyme in the metabolism of triglycerides such as those found in chylomicrons and very low-density lipoproteins (VLDL), and also promotes cellular uptake of chylomicron remnants, cholesterol-rich lipoproteins, and free fatty acids. LPL is synthesized and secreted by myocytes and adipocytes, but is transported to the lumen of capillaries, where it hydrolyses lipoprotein triglycerides. However, until recently there has been uncertainty as to how LPL is transported across the lumen wall.

Professor Young discussed recent findings showing that a GPI-anchored endothelial cell protein, GPIHBP1, plays a key role. Interestingly, GPIHBP1 is a member of the protein family typically found in snake venoms, known as three-finger proteins, characterised by Ly6 and acidic domains. These domains are key to the binding of these proteins and resultant effects on nerve junctions such as paralysis.

In recent years, it was shown that mammals also have this class of protein. Most of these proteins are anchored to the cell surface by a GPI anchor. Interest was focused on GPIHBP1 after the discovery that mice deficient in GPIHPB1 have severe hypertriglyceridemia and chylomicronemia, even on standard chow diets (with levels >3000 mg/dL). Experimental studies showed that the expression pattern of GPIHPB1 and LPL was very similar and also similar to CD31, the fatty acid transporter. However, there are differences; CD31 is expressed in all endothelial cells whereas in the case of GPIHPB1 it is expressed exclusively in the smallest capillaries. As the size of the capillaries increases by 50%, GPIHPB1 is absent. ‘Clearly GPIHPB1 is required for the transport of LPL from the basolateral to apical surface of endothelial cells.’

A number of missense mutations of GPIHPB1 have been identified in humans and are associated with substantially elevated triglyceride levels. Structural studies have shown that several involve mutations in the domains of this protein important in defining the three-fingered structure of this protein. In these mutations, the ability of the protein to bind LPL was abolished. These data indicate that there is more than one region of the GPIHPB1 protein that is important for binding LPL.

In electron microscopy studies, it was shown that GPIHPB1 is located exclusively in the caveolae of endothelial cells: ‘We imagine that chylomicrons sit like a bottle cap on top of the caveolae where they are subjected to the action of LPL. It is also possible that GPIHBP1 may influence triglyceride metabolism in other ways, aside from its transport function.’

The gut microbiota: an environmental factor that requires host physiology and metabolism

Professor Fredrik Bäckhed, Sahlgrenska Center for Cardiovascular and Metabolic Research/Wallenberg Laboratory, Department of Molecular and Clinical Medicine, University of Gothenburg, Sweden

In an interesting presentation, Professor Fredrik Bäckhed overviewed evidence for the human gut microbiota as an environmental factor influencing obesity. Studies show that germ-free mice, raised in the absence of exposure to bacteria, had 40% less total body fat than normal mice, even if their calorie intake was almost one-third higher. Additionally, when germ-free mice were fed a high-fat, high-carbohydrate Western diet, they were protected against diet-induced obesity, glucose intolerance and insulin resistance. ‘Two-way interaction between the gut microbiota, diet and host genotype may impact the metabolic phenotype.’

The mechanism(s) of these effects has been the subject of much investigation. Professor Bäckhed explained that an inhibitor of adipose tissue lipoprotein lipase, fasting-induced adipose factor (FIAF, also known as angiopoietin-like protein 4) might be key. The expression of FIAF was increased in germ-free mice. Additionally, activation of hepatic and muscle fatty acid oxidative pathways was also increased. Together, these data suggest that the microbiota may modulate the host metabolism.

This may occur via several mechanisms. These include an effect on host lipid metabolism, in turn modulating the expression of genes involved in bile acid secretion, indirectly influencing hepatic fat storage through bile acid signalling properties. Additionally, there may be effects mediated by low-grade endotoxins from the bacteria which counteract inflammation. Metabolic pathways are functionally integrated with immune responses, implying the relevance of the innate immune system for the pathogenesis of metabolic disorders.

Very recent data show that dietary supplementation of mice with phosphatidyl choline led to upregulation of multiple macrophage scavenger receptors which protected against atherosclerosis. Studies using germ-free mice confirmed a critical role for dietary choline and gut flora in augmenting macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary-choline-enhanced atherosclerosis.

Thus the gut microbiota can be considered an environmental factor that modulates obesity and other metabolic diseases. This may provide a rationale for a personalised approach via effects on the gut microbiota and opportunities for new therapeutic approaches for preventing atherosclerotic heart disease.

The diversity of HDL particles: a challenge for treatment

Professor M. John Chapman, Pitié-Salpétrière University Hospital, INSERM UMR-S939, Paris, France

The last decade has seen an explosion of information relating to the quality of HDL particles, especially in terms of their relevance to HDL atheroprotective function. HDL play a key role in maintaining cholesterol homeostasis via cellular cholesterol efflux, and display a spectrum of other potentially relevant atheroprotective, vasculoprotective and antithrombogenic activities. Indeed, a recent study showed that the capacity of HDL to promote cellular cholesterol efflux was independently predictive of a decrease in CIMT and risk of coronary artery disease.

On a physico-chemical basis, there are five major subpopulations of HDL particles, with apoA-I as the basic building block. The protein clusters on HDL may be a key determinant of its functionality. Indeed, the functional significance of HDL subpopulations reflects the integrated biological activity of both lipid (lipidome) and protein (proteome) components.

The question arises as to whether HDL structure and function might be relevant to cardiometabolic disease, especially when it features the atherogenic dyslipidemia of elevated triglyceride-rich lipoproteins and low levels of HDL cholesterol. Cholesteryl ester transfer protein (CETP) plays a key role in driving the production of small, triglyceride-rich HDL on a background of elevated CETP activity. The associated changes in the proteome are in part due to the enrichment of SAA, oxidation of amino acid residues in association with oxidative stress, and glycation of apoA-I; the lipidome may equally be altered by oxidation, lipid transfer activity and altered lipase activity. Such particles are functionally defective in cardiometabolic disease; however, it is important to stress that they do not exhibit a complete lack of function and neither are they pro-inflammatory.

What effects do pharmacological treatments that raise HDL cholesterol have on HDL structure and functionality? Although statins have a modest effect in raising HDL cholesterol, this has been shown to be critical in reducing plaque volume (together with lowering LDL cholesterol). Studies have shown that statins mainly shift the spectrum of HDL particles towards HDL2 with reduction in smaller particles, enhanced lipidation and prolonged residence time of apoA-I. In part, this is due to the effect of statin in reducing the numbers and concentration of acceptor particles such as VLDL and LDL for CETP-mediated transfer of cholesteryl ester from HDL. Reduction in the mass of CETP equally occurs and is intimately related to the LDL lowering efficacy of statins. The greater the reduction in acceptor particles, the greater the increase in HDL2 levels. In contrast, fibrates exert variable effects, and vary in their potential to raise HDL levels and this effect is related to the metabolic background. Indeed, on a background of diabetes/insulin resistance induction of homocysteine attenuates the increase in HDL via impact on apoA-I, typically increasing HDL-C levels by <5%. In the case of niacin, there is a more marked increase in larger HDL particles compared with either statins or fibrates, and decrease in small pre beta HDL particles. Niacin is a strong driver of apo-AI production; niacin equally mediates an increase in HDL-C levels via its impact on CETP, as it may decrease mass and activity by up to 30%. Thus, CETP is at the heart of the lipid modulating action of niacin and statins.

Professor Chapman highlighted unmet challenges in the field of HDL particle structure and function. Four are key: