Distinct Roles of Apolipoproteins A1 and E in the Modulation of High-Density Lipoprotein Composition and Function.

In addition to high-density lipoprotein cholesterol (HDL-C) levels, HDL quality also appears to be very important for atheroprotection. Analysis of various clinical paradigms suggests that the lipid and apolipoprotein composition of HDL defines its size, shape, and functions and may determine its beneficial effects on human health. Previously, we reported that like apolipoprotein A-I (Apoa1), apolipoprotein E (Apoe) is also capable of promoting the de novo biogenesis of HDL with the participation of ATP binding cassette A lipid transporter member 1 (Abca1) and plasma enzyme lecithin:cholesterol acyltransferase (Lcat), in a manner independent of a functional Apoa1. Here, we performed a comparative analysis of the functions of these HDL subpopulations. Specifically, Apoe and Apoa1 double-deficient (Apoe(-/-) × Apoa1(-/-)) mice were infected with APOA1- or APOE3-expressing adenoviruses, and APOA1-containing HDL (APOA1-HDL) and APOE3-containing HDL (APOE3-HDL), respectively, were isolated and analyzed by biochemical and physicochemical methods. Western blot and lipidomic analyses indicated significant differences in the apolipoprotein and lipid composition of the two HDL species. Moreover APOE3-HDL presented a markedly reduced antioxidant potential and Abcg1-mediated cholesterol efflux capacity. Surprisingly, APOE3-HDL but not APOA1-HDL attenuated LPS-induced production of TNFα in RAW264.7 cells, suggesting that the anti-inflammatory effects of APOA1 are dependent on APOE expression. Taken together, our data indicate that APOA1 and APOE3 recruit different apolipoproteins and lipids on the HDL particle, leading to structurally and functionally distinct HDL subpopulations. The distinct role of these two apolipoproteins in the modulation of HDL functionality may pave the way toward the development of novel pharmaceuticals that aim to improve HDL functionality.

Lack of LCAT reduces the LPS-neutralizing capacity of HDL and enhances LPS-induced inflammation in mice.

HDL has important immunomodulatory properties, including the attenuation of lipopolysaccharide (LPS)-induced inflammatory response. As lecithin-cholesterol acyltransferase (LCAT) is a critical enzyme in the maturation of HDL we investigated whether LCAT-deficient (Lcat(-/-)) mice present an increased LPS-induced inflammatory response. LPS (100µg/kg body weight)-induced cytokine response in Lcat(-/-) mice was markedly enhanced and prolonged compared to wild-type mice. Importantly, reintroducing LCAT expression using adenovirus-mediated gene transfer reverted their phenotype to that of wild-type mice. Ex vivo stimulation of whole blood with LPS (1-100ng/mL) showed a similar enhanced pro-inflammatory phenotype. Further characterization in RAW 264.7 macrophages in vitro showed that serum and HDL, but not chylomicrons, VLDL or the lipid-free protein fraction of Lcat(-/-) mice, had a reduced capacity to attenuate the LPS-induced TNFα response. Analysis of apolipoprotein composition revealed that LCAT-deficient HDL lacks significant amounts of ApoA-I and ApoA-II and is primarily composed of ApoE, while HDL from Apoa1(-/-) mice is highly enriched in ApoE and ApoA-II. ApoA-I-deficiency did not affect the capacity of HDL to neutralize LPS, though Apoa1(-/-) mice showed a pronounced LPS-induced cytokine response. Additional immunophenotyping showed that Lcat(-/-) , but not Apoa1(-/-) mice, have markedly increased circulating monocyte numbers as a result of increased Cd11b(+)Ly6C(med) monocytes, whereas 'pro-inflammatory' Cd11b(+)Ly6C(hi) monocytes were reduced. In line with this observation, peritoneal macrophages of Lcat(-/-) mice showed a markedly dampened LPS-induced TNFα response. We conclude that LCAT-deficiency increases LPS-induced inflammation in mice due to reduced LPS-neutralizing capacity of immature discoidal HDL and increased monocyte number.

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.

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.

The low density lipoprotein receptor modulates the effects of hypogonadism on diet-induced obesity and related metabolic perturbations.

Here, we investigated how LDL receptor deficiency (Ldlr(-/-)) modulates the effects of testosterone on obesity and related metabolic dysfunctions. Though sham-operated Ldlr(-/-) mice fed Western-type diet for 12 weeks became obese and showed disturbed plasma glucose metabolism and plasma cholesterol and TG profiles, castrated mice were resistant to diet-induced obesity and had improved glucose metabolism and reduced plasma TG levels, despite a further deterioration in their plasma cholesterol profile. The effect of hypogonadism on diet-induced weight gain of Ldlr(-/-) mice was independent of ApoE and Lrp1. Indirect calorimetry analysis indicated that hypogonadism in Ldlr(-/-) mice was associated with increased metabolic rate. Indeed, mitochondrial cytochrome c and uncoupling protein 1 expression were elevated, primarily in white adipose tissue, confirming increased mitochondrial metabolic activity due to thermogenesis. Testosterone replacement in castrated Ldlr(-/-) mice for a period of 8 weeks promoted diet-induced obesity, indicating a direct role of testosterone in the observed phenotype. Treatment of sham-operated Ldlr(-/-) mice with the aromatase inhibitor exemestane for 8 weeks showed that the obesity of castrated Ldlr(-/-) mice is independent of estrogens. Overall, our data reveal a novel role of Ldlr as functional modulator of metabolic alterations associated with hypogonadism.

Whole-Slide Image Analysis of Human Pancreas Samples to Elucidate the Immunopathogenesis of Type 1 Diabetes Using the QuPath Software.

Type 1 diabetes is a chronic disease of the pancreas characterized by the loss of insulin-producing beta cells. Access to human pancreas samples for research purposes has been historically limited, restricting pathological analyses to animal models. However, intrinsic differences between animals and humans have made clinical translation very challenging. Recently, human pancreas samples have become available through several biobanks worldwide, and this has opened numerous opportunities for scientific discovery. In addition, the use of new imaging technologies has unraveled many mysteries of the human pancreas not merely in the presence of disease, but also in physiological conditions. Nowadays, multiplex immunofluorescence protocols as well as sophisticated image analysis tools can be employed. Here, we described the use of QuPath-an open-source platform for image analysis-for the investigation of human pancreas samples. We demonstrate that QuPath can be adequately used to analyze whole-slide images with the aim of identifying the islets of Langerhans and define their cellular composition as well as other basic morphological characteristics. In addition, we show that QuPath can identify immune cell populations in the exocrine tissue and islets of Langerhans, accurately localizing and quantifying immune infiltrates in the pancreas. Therefore, we present a tool and analysis pipeline that allows for the accurate characterization of the human pancreas, enabling the study of the anatomical and physiological changes underlying pancreatic diseases such as type 1 diabetes. The standardization and implementation of these analysis tools is of critical importance to understand disease pathogenesis, and may be informative for the design of new therapies aimed at preserving beta cell function and halting the inflammation caused by the immune attack.

Lipocalin-2 counteracts metabolic dysregulation in obesity and diabetes.

Regulation of food intake is a recently identified endocrine function of bone that is mediated by Lipocalin-2 (LCN2). Osteoblast-secreted LCN2 suppresses appetite and decreases fat mass while improving glucose metabolism. We now show that serum LCN2 levels correlate with insulin levels and ß-cell function, indices of healthy glucose metabolism, in obese mice and obese, prediabetic women. However, LCN2 serum levels also correlate with body mass index and insulin resistance in the same individuals and are increased in obese mice. To dissect this apparent discrepancy, we modulated LCN2 levels in mice. Silencing Lcn2 expression worsens metabolic dysfunction in genetic and diet-induced obese mice. Conversely, increasing circulating LCN2 levels improves metabolic parameters and promotes ß-cell function in mouse models of ß-cell failure acting as a growth factor necessary for ß-cell adaptation to higher metabolic load. These results indicate that LCN2 up-regulation is a protective mechanism to counteract obesity-induced glucose intolerance by decreasing food intake and promoting adaptive ß-cell proliferation.

Lipocalin-2 is an anorexigenic signal in primates.

In the mouse, the osteoblast-derived hormone Lipocalin-2 (LCN2) suppresses food intake and acts as a satiety signal. We show here that meal challenges increase serum LCN2 levels in persons with normal or overweight, but not in individuals with obesity. Postprandial LCN2 serum levels correlate inversely with hunger sensation in challenged subjects. We further show through brain PET scans of monkeys injected with radiolabeled recombinant human LCN2 (rh-LCN2) and autoradiography in baboon, macaque, and human brain sections, that LCN2 crosses the blood-brain barrier and localizes to the hypothalamus in primates. In addition, daily treatment of lean monkeys with rh-LCN2 decreases food intake by 21%, without overt side effects. These studies demonstrate the biology of LCN2 as a satiety factor and indicator and anorexigenic signal in primates. Failure to stimulate postprandial LCN2 in individuals with obesity may contribute to metabolic dysregulation, suggesting that LCN2 may be a novel target for obesity treatment.

Obesity has reached epidemic proportions worldwide and affects more than 40% of adults in the United States. People with obesity have a greater likelihood of developing type 2 diabetes, cardiovascular disease or chronic kidney disease. Changes in diet and exercise can be difficult to follow and result in minimal weight loss that is rarely sustained overtime. In fact, in people with obesity, weight loss can lower the metabolism leading to increased weight gain. New drugs may help some individuals achieve 5 to 10% weight loss but have side effects that prevent long-term use. Previous studies in mice show that a hormone called Lipocalin-2 (LCN2) suppresses appetite. It also reduces body weight and improves sugar metabolism in the animals. But whether this hormone has the same effects in humans or other primates is unclear. If it does, LCN2 might be a potential obesity treatment. Now, Petropoulou et al. show that LCN2 suppressed appetite in humans and monkeys. In human studies, LCN2 levels increased after a meal in individuals with normal weight or overweight, but not in individuals with obesity. Higher levels of LCN2 in a person's blood were also associated with a feeling of reduced hunger. Using brain scans, Petropoulou et al. showed that LCN2 crossed the blood-brain barrier in monkeys and bound to the hypothalamus, the brain center regulating appetite and energy balance. LCN2 also bound to human and monkey hypothalamus tissue in laboratory experiments. When injected into monkeys, the hormone suppressed food intake and lowered body weight without toxic effects in short-term studies. The experiments lay the initial groundwork for testing whether LCN2 might be a useful treatment for obesity. More studies in animals will help scientists understand how LCN2 works, which patients might benefit, how it would be given to patients and for how long. Clinical trials would also be needed to verify whether it is an effective and safe treatment for obesity.

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.

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.

Effects of bariatric surgery on HDL structure and functionality: results from a prospective trial.

BACKGROUND: In addition to high-density lipoprotein cholesterol (HDL-C) levels, HDL quality appears also very important for atheroprotection. Obese patients with metabolic syndrome have significantly reduced HDL-C levels and are usually at increased risk for coronary heart disease. Despite that weight loss benefits these patients, its effects on HDL quality and functionality is currently poorly studied. OBJECTIVES: We investigated how rapid weight loss affects HDL structure and its antioxidant potential in patients undergoing a malabsorptive bariatric procedure. METHODS: Fasting plasma samples were collected the day before and 6 months after the bariatric procedure from 20 morbidly obese patients with body mass index >50, then HDL was isolated and analyzed by biochemical techniques. RESULTS: We report a dramatic alteration in the apolipoprotein ratio of HDL that was accompanied by the presence of more mature HDL subspecies and a concomitant increase in the antioxidant potential of HDL. Interestingly, our obese cohort could be distinguished into 2 subgroups. In 35% of patients (n = 7), HDL before surgery had barely detectable apolipoprotein (apo) A-I and apoCIII, and the vast majority of their HDL cholesterol was packed in apoE-containing HDL particles. In the remaining 65% of patients (n = 13), HDL before surgery contained high levels of apoA-I and apoCIII, in addition to apoE. In both subgroups, surgical weight loss resulted in a switch from apoE to apoA-I-containing HDL. CONCLUSIONS: Rapid weight loss exerts a significant improvement in HDL structure and functionality that may contribute to the documented beneficial effect of malabsorptive bariatric procedures on cardiovascular health.

Regulation of endothelial nitric oxide synthase and high-density lipoprotein quality by estradiol in cardiovascular pathology.

Estrogens have been recognized, in the last 3 decades, as important hormones in direct and indirect modulation of vascular health. In addition to their direct benefit on cardiovascular health, the presence of esterified estrogen in the lipid core of high-density lipoprotein (HDL) particles indirectly contributes to atheroprotection by significantly improving HDL quality and functionality. Estrogens modulate their physiological activity via genomic and nongenomic mechanisms. Genomic mechanisms are thought to be mediated directly by interaction of the hormone receptor complex with the hormone response elements that regulate gene expression. Nongenomic mechanisms are thought to occur via interaction of the estrogen with membrane-bound receptors, which rapidly activate intracellular signaling without binding of the hormone receptor complex to its hormone response elements. Estradiol in particular mediates early and late endothelial nitric oxide synthase (eNOS) activation via interaction with estrogen receptors through both nongenomic and genomic mechanisms. In the vascular system, the primary endogenous source of nitric oxide (NO) generation is eNOS. Nitric oxide primarily influences blood vessel relaxation, the heart rate, and myocyte contractility. The abnormalities in expression and/or functions of eNOS lead to the development of cardiovascular diseases, both in animals and in humans. Although considerable research efforts have been dedicated to understanding the mechanisms of action of estradiol in regulating cardiac eNOS, more research is needed to fully understand the details of such mechanisms. This review focuses on recent findings from animal and human studies on the regulation of eNOS and HDL quality by estradiol in cardiovascular pathology.