Katherine Cianflone

Hypertriglyceridemic hyperapoB: The unappreciated atherogenic dyslipoproteinemia in type 2 diabetes mellitus

Worldwide, more than 100 million people have type 2 diabetes mellitus, and that number will more than double in the next 10 years (1). Of these persons, more than three of fourthat is, more than 150 million peopleare expected to die of cardiovascular disease over the next decade (2). A history of diabetes is equivalent in risk for death to a history of myocardial infarction, and the combination compounds the risk (3). For some time, the hope has been that better control of glucose would substantially reduce the risk for vascular disease. To date, however, that has not been the case. The United Kingdom Prospective Diabetes Study found that better control reduced the frequency of microvascular disease, but the trend toward a reduced frequency of macrovascular disease was not statistically significant (4). In addition, the frequency of macrovascular disease in patients with type 2 diabetes varies geographically (5), suggesting that factors other than diabetes play an important role in the pathogenesis of the vascular disease. One such factor may be the dyslipoproteinemias that are common in diabetic patients. This review focuses on hypertriglyceridemic hyperapoB: the combination of hypertriglyceridemia; increased numbers of small, dense low-density lipoprotein (LDL) particles; and, often, low levels of high-density lipoprotein (HDL) cholesterol. This atherogenic lipoprotein profile is not restricted to persons with type 2 diabetes mellitus (6, 7); it is also common in persons with insulin resistance (8-11), those who will develop diabetes (12-15), and those with coronary disease (16-18). Accordingly, we believe that physicians should understand its pathophysiology in order to better select and monitor therapy. Lipoprotein Particles and Lipoprotein Lipids Clinical practice has been based on plasma concentrations of the major lipoprotein lipids triglycerides and cholesterol. Both substances are insoluble in water and therefore must circulate in plasma within lipoprotein particles. Because very-low-density lipoproteins (VLDLs) are much larger than LDL particles, the distinction between the concentration of lipids in plasma and the concentration of lipoprotein particles in plasma is critical. Figure 1 illustrates this principle in a patient with a plasma triglyceride level of 3 mmol/L (264 mg/dL) and an LDL cholesterol level of 3 mmol/L (116 mg/dL). The cholesterol is mainly present within LDL particles, and triglyceride is mainly found within VLDL particles. In this example, the plasma concentrations of triglyceride and LDL cholesterol are the same as measured in mmol/L, but the triglyceride concentration is twice as great when measured in mg/dL. Nevertheless, because LDL particles are much smaller than VLDL particles, there are always many more LDL particles than VLDL particles per volume of plasma. Figure 1. Differences between lipoprotein lipids and lipoprotein particles in a patient with a plasma triglyceride level of 3 mmol/L (264 mg/dL) and a low-density lipoprotein ( LDL ) cholesterol level of 3 mmol/L (116 mg/dL). The hepatic apoB lipoproteins are shown in Figure 2. They include VLDL particles; intermediate-density lipoprotein particles, which are produced by the partial hydrolysis of triglycerides within VLDL and are therefore smaller because they contain fewer triglycerides; and LDL particles, which are the end product of VLDL metabolism. Very-low-density lipoprotein, intermediate-density lipoprotein, and LDL are spherical particles covered by a phospholipid monolayer, within which are intercalated molecules of free cholesterol and a single molecule of a very large protein, apoB100 (19) (Figure 2). The molecule of apoB100 provides structural integrity for the particle and remains part of it for the particles entire lifetime. ApoB100 binds to the LDL receptor and is a crucial link in the normal pathway by which LDL is removed from plasma (20). Numerous other proteins are also present in the monolayer of a VLDL particle but are gradually shed as VLDL is converted to LDL. The one molecule of the apoB100 is the only protein in the LDL external monolayer. Triglycerides and cholesterol ester are the two lipids within the core of the particle, but their absolute and relative amounts differ in the various apoB lipoproteins. In VLDL, most of the core lipids are triglycerides; intermediate-density lipoproteins are almost half triglycerides and half cholesterol esters; and LDL is mainly composed of cholesterol ester. Figure 2. The relative number of very-low-density lipoprotein (VLDL) ( left ), intermediate-density lipoprotein ( middle ), and low-density lipoprotein (LDL) ( right ) particles. Because each hepatic apoB lipoprotein secreted by the liver contains one molecule of apoB100, the concentration of apoB in plasma measures the total number of VLDL, intermediate-density lipoprotein, and LDL lipoprotein particles in plasma (19). Lipoprotein(a) also contains one molecule of apoB but characteristically does not contribute substantially to total apoB in hyperapoB (21). Most LDL particles are metabolic products of VLDL particles, but their half-lives differ greatly because LDL particles persist in the circulation for about nine times longer than do VLDL particles. This explains why there are nine times more LDL particles than VLDL particles, even in hypertriglyceridemic patients (22) (Figure 2). For clinical purposes, therefore, the number of LDL particles can be inferred from the plasma apoB concentration. Measurement of apoB is now standardized and automated (23-25), and apoB can be accurately measured in clinical laboratories. Reference values have been determined (26-28) and fasting samples are not required (29), features that are especially important in diabetic patients. Small, Dense LDL Particles Not only does the composition of the various apoB lipoproteins differ, but the composition of particles within a particular class can differ importantly. In the case of LDL particles, some are larger and more buoyant (LDL A), whereas others are smaller and denser (LDL B) (30) (Figure 2). The LDL A particles contain more cholesterol ester per particle than do the LDL B particles. Most LDL B particles are formed from LDL A particles (31); Figure 3 shows the mechanisms responsible for this transformation. Figure 3. Formation of small, dense low-density lipoprotein ( LDL ) particles. CETP LPL HL Originally, it was thought that the core lipids cholesterol ester and triglyceride were fixed components of the particles and could not be exchanged among them. However, Morton and Zilversmits (32) description of cholesterol ester transfer protein changed this viewpoint. If a triglyceride molecule is exchanged for another triglyceride molecule and a cholesterol ester is exchanged for another cholesterol ester, the core lipid composition will remain the same. In contrast, if a triglyceride from VLDL is exchanged for a cholesterol ester in LDL, the core lipid composition of both lipoproteins will change: The VLDL becomes enriched in cholesterol ester, and LDL becomes enriched in triglyceride (Figure 3). This first step does not change their size or density. However, if the triglyceride in LDL is hydrolyzed by lipoprotein lipase or hepatic lipase, a smaller, denser LDL particle will be produced. The rate at which this remodeling occurs depends on the triglyceride content of the donor particle (33) and the number of donor and recipient particles. Therefore, increased VLDL secretion results in increased generation of small, dense LDL particles (34)that is, it causes hypertriglyceridemic hyperapoB (Figure 4). Increased plasma fatty acid levels also increase LDL remodeling (35). On the other hand, abnormalities in glucose metabolism alone do not seem to influence the process, although the evidence is mixed (36, 37). The size of LDL particles cannot be measured directly in routine clinical laboratories, but the relationship between plasma triglyceride and small, dense LDL is predictable. Thus, when triglyceride concentrations are less than 1.5 mmol/L (132 mg/dL), small, dense LDL particles are unlikely to be present, but at triglyceride concentrations of 1.5 mmol/L or greater, and particularly greater than 2.0 mmol/L (176 mg/dL), the opposite is the case (17, 38, 39). Figure 4. ApoB lipoprotein particles in healthy persons ( left ) and those with hypertriglyceridemic hyperapoB ( right ). A low HDL cholesterol level is very common in patients with coronary disease (40). It is often caused by the core lipid exchanges described above, but in this instance, they also lead to accelerated clearance of HDL particles. These interactions explain why hypertriglyceridemia and low HDL cholesterol levels so frequently coexist. Atherogenic Risks of Hypertriglyceridemic HyperapoB Hypertriglyceridemia and low HDL cholesterol levels are much more common in patients with coronary disease than are elevations in total and LDL cholesterol levels (41); thus, from an epidemiologic perspective, both are important risk factors for premature vascular disease. The HDL cholesterol level appears to be the more important factor; both univariate and multivariate analyses usually show it to be a significant risk factor, whereas hypertriglyceridemia is often not significant on multivariate analyses. However, the importance of this should not be overstated, particularly given the role of triglyceride in lipoprotein remodeling and, therefore, in producing low HDL cholesterol levels (42, 43). A low HDL cholesterol level was originally thought to result from impaired reverse transport of cholesterol from the periphery to the liver (44). This is not invariably the case, however; for example, genetic mutations that result in a low HDL cholesterol level are not always associated with an increased risk for vascular disease (45). High-density lipoprotein cholesterol may have other roles, including inhibition of LDL oxidation and expression of endothelial adhesion molecules (46

The chemoattractant receptor-like protein C5L2 binds the C3a des-Arg77/acylation-stimulating protein

The orphan receptor C5L2 has recently been described as a high affinity binding protein for complement fragments C5a and C3a that, unlike the previously described C5a receptor (CD88), couples only weakly to Gi-like G proteins (Cain, S. A., and Monk, P. N. (2002) J. Biol. Chem. 277, 7165–7169). Here we demonstrate that C5L2 binds the metabolites of C4a and C3a, C4a des-Arg77, and C3a des-Arg77 (also known as the acylation-stimulating protein or ASP) at a site distinct from the C5a binding site. The binding of these metabolites to C5L2 does not stimulate the degranulation of transfected rat basophilic leukemia cells either through endogenous rat G proteins or when co-transfected with human Gα16. C3a des-Arg77/ASP and C3a can potently stimulate triglyceride synthesis in human skin fibroblasts and 3T3-L1 preadipocytes. Here we show that both cell types and human adipose tissue express C5L2 mRNA and that the human fibroblasts express C5L2 protein at the cell surface. This is the first demonstration of the expression of C5L2 in cells that bind and respond to C3a des-Arg77/ASP and C3a. Thus C5L2, a promiscuous complement fragment-binding protein with a high affinity site that binds C3a des-Arg77/ASP, may mediate the acylation-stimulating properties of this peptide.

C5L2 is a functional receptor for acylation-stimulating protein.

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 ± 33%, 5 μm ASP, p < 0.001, where basal = 100%) and glucose transport (168 ± 21%, 10 μm ASP, p < 0.001). C3a similarly stimulates TGS (163 ± 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 ± 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 ± 1.6, with ASP = 21.0 ± 1.4 (144%), with ASP + oligonucleotides = 11.0 ± 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 ± 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 ± 2.9, with ASP = 39.6 ± 8.8 (177%), with ASP + oligonucleotides = 25.3 ± 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged β-arrestin, ASP induced β-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.

Acylation Stimulating Protein (ASP) Deficiency Alters Postprandial and Adipose Tissue Metabolism in Male Mice

Acylation stimulating protein (ASP) is a potent stimulator of triglyceride synthesis in adipocytes. In the present study, we have examined the effect of an ASP functional knockout (ASP(−/−)) on lipid metabolism in male mice. In both young (14 weeks) and older (26 weeks) mice there were marked delays in postprandial triglyceride clearance (80% increase at 14 weeks and 120% increase at 26 weeks versus wild type (+/+)). Postprandial nonesterified fatty acids were also increased in ASP(−/−) miceversus ASP(+/+) mice by 37% (low fat 10% Kcal) and by 73% (high fat 40% Kcal) diets, although there were no differences in fasting lipid levels. The ASP(−/−) mice had moderately increased energy intake (16% ± 2% p < 0.0001) and reduced feed efficiency (33% increase in calories/g of body weight gained on low fat diet) versus wild type. The ASP(−/−) mice also had modest changes in insulin/glucose metabolism (30% to 40% decrease in insulin·glucose product), implying increased insulin sensitivity. As well, there were decreases in leptin (29% shift in leptin to body weight ratio) and up to a 26% decrease in specific adipose tissue depots versus the wild type mice on both low fat and high fat diets. These results demonstrate that ASP plays an important role in adipose tissue metabolism and fat partitioning.

Leptin and adiponectin in relation to body fat percentage, waist to hip ratio and the apoB/apoA1 ratio in Asian Indian and Caucasian men and women

BackgroundAsian Indian immigrants have an increased risk for developing cardiovascular disease (CVD); however, there is very little data examining how the adipokines leptin and adiponectin relate to CVD risk factors such as body fat percentage (BF%), waist to hip ratio (WHR) and the apoB/apoA1 ratio in Asian Indian men and women living in Canada.Subjects and methodsA cross-sectional study comparing leptin, adiponectin, lipoproteins and anthropometric parameters in Asian Indian men and women to Caucasian men and women (4 groups). Anthropometric data (BMI, BF%, WHR), circulating lipids (apoA1, apoB, total cholesterol, and HDL-cholesterol), leptin and adiponectin were measured.ResultsAsian Indian men and women had higher leptin and lower adiponectin concentrations then Caucasian men and women, respectively. Leptin (positively) and adiponectin (negatively) correlated with anthropometric parameters and lipoproteins in all four groups. Using stepwise forward multiple regression, a model including TC/HDL-C ratio, WHR, BF%, hip circumference and waist circumference predicted 74.2% of leptin concentration in men. In women, apoB, BF%, waist circumference and age predicted 77.5% of leptin concentration. Adiponectin concentrations in men were predicted (30.2%) by HDL-C, total cholesterol, hip circumference and BF% while in women 41.2% of adiponectin concentration was predicted by the apoB/apoA1 ratio, WHR and age.ConclusionAs is evident from our data, there is a strong relationship between leptin, adiponectin, and abdominal obesity with increased CVD risk, as assessed by the apoB/apoA1 ratio. Dysregulation of these parameters may account for the increased risk of CVD in Asian Indians.

Regulation of plasma fatty acid metabolism

Although adipose tissue serves a crucial function in energy storage, excess adipose tissue--that is, obesity--is often associated with diabetes and cardiovascular disease. A common thread in the weave of complications is increased plasma concentrations of fatty acids. In the present review, we have focused on two specific points that relate to obesity: (i) What are the metabolic consequences of increased free fatty acid concentrations? and (ii) What are the physiological factors that are involved in the regulation of fatty acid uptake or release from adipose tissue? We have tried to emphasize new factors that act as hormones on adipose tissue and in so doing regulate the net concentration of circulating free fatty acids.

Novel roles for acylation stimulating protein/C3adesarg : A review of recent in vitro and in vivo evidence

Recent experimental evidence is shedding more light on the physiological actions of acylation-stimulating protein (ASP)/C3adesArg. The role of ASP in regulating lipid metabolism has primarily focused on its participation in the stimulation of triglyceride synthesis (TGS) and glucose transport. Although there is no doubt that ASP, an adipocyte-produced hormone, plays a key physiological role, accumulating evidence suggests that the effects of ASP go beyond its acute effects on lipid metabolism. In this review, we present novel findings of ASP/C3adesArg effects on preadipocyte differentiation. In 3T3-L1 and 3T3-F442A cells, ASP can substitute for insulin and enhance differentiation as measured by intracellular lipid droplet accumulation, clonal expansion, and increased expression of differentiation markers. Specifically, ASP increased basal TGS by 250% after 9 days differentiation, with similar effects induced by insulin. With ASP treatment, expression of C/EBPdelta was up-regulated early in differentiation (day 2) and decreased thereafter. Expression of PPARgamma and late markers of differentiation, such as adipsin and diacylglycerol acyltransferase-1, were also increased. Effects on clonal expansion were indicated by a twofold increase in [(3)H] thymidine incorporation in 3T3-L1 cells compared to treatment with IBMX + DX alone. Further, the effects of ASP extended beyond adipose tissue to endocrine effects on hormone secretion of insulin (pancreatic cells); cytokines TNFalpha, IL-1beta, and IL-6 (myeloid cells); prolactin, growth hormone, and adrenocorticotropin (pituitary cells). Finally, the potential implication of C5L2, the newly discovered ASP receptor, and its expression profile in various tissues are discussed relative to ASP function.

Enhanced triglyceride clearance with intraperitoneal human acylation stimulating protein in C57BL/6 mice.

Acylation stimulating protein (ASP), a novel adipocyte-derived autocrine protein, stimulates triglyceride synthesis and glucose transport in vitro in human and murine adipocytes. In vitro, chylomicrons increase ASP and precursor complement C3 production in adipocytes. Furthermore, in vivo, ASP production from human adipose tissue correlates positively with triglyceride clearance postprandially. The aim of the present study was to determine if intraperitoneally injected ASP accelerated triglyceride clearance in vivo after a fat load in C57Bl/6 mice. ASP increased the triglyceride clearance with a reduction of the triglyceride area under the curve over 6 h (AUC0-6) from 102.6 ± 30.0 to 61.0 ± 14.5 mg ⋅ dl-1 ⋅ h-1( P < 0.05), especially in the latter postprandial period (AUC3-6; 56.2 ± 18.0 vs. 24.9 ± 8.9 mg ⋅ dl-1 ⋅ h-1, P < 0.025). ASP also reduced plasma glucose both in the mice with accelerated plasma triglyceride clearance and in those with relatively delayed triglyceride clearance ( P < 0.025). Therefore, ASP alters postprandial triglyceride and glucose metabolism.

Governance of the concentration of plasma LDL: a reevaluation of the LDL receptor paradigm

The level of LDL in plasma is the major determinantof the risk of vascular disease and lowering the level ofLDL diminishes that risk, both in those with and thosewithout symptomatic vascular disease [1–4]. Fully un-derstanding the factors that govern the concentration ofplasma LDL is, therefore, one of our most importantchallenges. Since its enunciation [5,6], the LDL receptorparadigm has dominated thinking in this area. In brief,it states that the major determinant of the concentra-tion of LDL is the rate at which LDL particles arecleared from plasma and that the rate of LDL clearancefrom plasma is determined by the activity of hepaticLDL receptors.The LDL receptor paradigm stipulates that choles-terol which has entered the cell via the LDL pathwayproduces three coordinated and concurrent events: de-creased cholesterol synthesis, decreased LDL receptorsynthesis, and increased cholesterol ester synthesis; allthree of which act to ensure the level of free cholesterolwithin the cell remains within the narrow limits neces-sary for normal membrane function and normal cellularfunction. On the other hand, LDL entering the cell byany non-LDL receptor pathway such as the scavengerreceptor will not elicit these self-limiting responses andaccordingly will lead to continuing unregulated accu-mulation of cholesterol within the cell. Thus one routeleads to synchronous self-correcting and therefore nor-mal responses, the other to progressive deviation anddisease. The LDL pathway is perhaps the best-knownexpression of biologic homeostasis in modern times.Two human metabolic disorders, familial hyperc-holesterolemia (FH) [7] and the defective apoB100 syn-drome [8], each of which has been explicitly defined atthe molecular level, testify unambiguously to the criticalbiologic importance of the LDL pathway for the hu-man organism. In the first, the LDL receptor is abnor-mal while in the second, the ligand for the LDLreceptor, apoB100, is abnormal. In both disorders, theclearance from plasma of LDL through the LDL path-way is markedly reduced, and consequently, in both,the levels of LDL are markedly elevated, as is the riskof coronary disease. The LDL receptor paradigmwould seem secure, and if it is, there is no need tosearch further to understand the regulation of plasmaLDL. But is it?Five years ago, Fisher, Zech and Stacpoole pointedout certain inconsistencies with the LDL receptorparadigm [9]. They noted that in WHHL rabbits, theanimal counterpart to FH [10], and in two humans withhomozygous (FH) [11], hepatic free cholesterol andcholesterol ester content were clearly although unex-pectedly increased. Moreover, in the WHHL rabbit,cholesterol synthesis is depressed [10] as is HMGCoAreductase activity and cholesterol synthesis in circulat-ing FH mononuclear cells [12]. These observations are,of course, inconsistent with the contention that onlycholesterol entering the cell via the LDL receptor canregulate internal cholesterol homeostasis [5,6].Since then, much more has been learned about theregulation of sterol balance within the hepatocyte, andby extension, within the organism as a whole. Thesedata lead to a model that is very different from theLDL paradigm: namely, that there is a second majordeterminant of the concentration of LDL in plasma —

Visfatin concentration in asian indians is correlated with high density lipoprotein cholesterol and apolipoprotein A1

Backgroundu2002 Visfatin is a recently described adipose tissue derived hormone whose role in humans remains largely unknown.