Cold acclimation induces physiological cardiac hypertrophy and increases assimilation of triacylglycerol metabolism through lipoprotein lipase

The contribution of triacylglycerol to energy provision in the hypertrophied heart, mediated through lipoprotein lipase (LPL) is largely unknown and the contribution of very-low-density lipoprotein (VLDL) receptor to control of LPL presentation at the endothelium is unclear. For isolated perfused rat hearts, cold acclimation (CA) induced volume-overload hypertrophy, with decreased developed pressure (P<0.01), increased end-diastolic volume of the left ventricle (P<0.001) and a loss of contractile reserve in response to dobutamine challenge (P<0.01). Oleate utilisation by perfused hearts was unchanged by CA, however uptake of intralipid emulsion increased 3-fold (P<0.01). CA increased the proportion of lipid deposited in tissue lipids from 10% in euthermic controls to 40% (P<0.01) although the overall contribution of individual lipid classes was unaffected. Cold acclimation significantly increased heparin-releasable LPL (P<0.05) and tissue residual LPL (P<0.01). Western blot analysis indicated preserved expression of proteins coding for SERCA2, muscle-CPT1 and VLDL-receptor following CA, while AMPKalpha2 and phospho-AMPKalpha2 were unaffected. These observations indicate that for physiological hypertrophy AMPK phosphorylation does not mediate the enhanced translocation of LPL to cardiac endothelium.     

Angiopoietin-like 4 Modifies the Interactions between Lipoprotein Lipase and Its Endothelial Cell Transporter GPIHBP1

The release of fatty acids from plasma triglycerides for tissue uptake is critically dependent on the enzyme lipoprotein lipase (LPL). Hydrolysis of plasma triglycerides by LPL can be disrupted by the protein angiopoietin-like 4 (ANGPTL4), and ANGPTL4 has been shown to inactivate LPL in vitro. However, in vivo LPL is often complexed to glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) on the surface of capillary endothelial cells. GPIHBP1 is responsible for trafficking LPL across capillary endothelial cells and anchors LPL to the capillary wall during lipolysis. How ANGPTL4 interacts with LPL in this context is not known. In this study, we investigated the interactions of ANGPTL4 with LPL-GPIHBP1 complexes on the surface of endothelial cells. We show that ANGPTL4 was capable of binding and inactivating LPL complexed to GPIHBP1 on the surface of endothelial cells. Once inactivated, LPL dissociated from GPIHBP1. We also show that ANGPTL4-inactivated LPL was incapable of binding GPIHBP1. ANGPTL4 was capable of binding, but not inactivating, LPL at 4 °C, suggesting that binding alone was not sufficient for ANGPTL4''s inhibitory activity. We observed that although the N-terminal coiled-coil domain of ANGPTL4 by itself and full-length ANGPTL4 both bound with similar affinities to LPL, the N-terminal fragment was more potent in inactivating both free and GPIHBP1-bound LPL. These results led us to conclude that ANGPTL4 can both bind and inactivate LPL complexed to GPIHBP1 and that inactivation of LPL by ANGPTL4 greatly reduces the affinity of LPL for GPIHBP1.     

The acute phase response stimulates the expression of angiopoietin like protein 4

The acute phase response is characterized by elevations in serum triglyceride levels due to both an increase in hepatic VLDL production and a delay in the clearance of triglyceride rich lipoproteins secondary to a decrease in lipoprotein lipase (LPL) activity. Recently there has been a marked increase in our understanding of factors that regulate LPL activity. GPIHBP1 facilitates the interaction of LPL and lipoproteins thereby allowing lipolysis to occur. Angiopoietin like proteins (ANGPTL) 3 and 4 inhibit LPL activity. In the present study, treatment of mice with LPS, an activator of TLR4 and a model of Gram-negative infections, did not alter the expression of GPIHBP1 in heart or adipose tissue. However, LPS decreased the expression of ANGPTL3 in liver and increased the expression of ANGPTL4 in heart, muscle, and adipose tissue. Serum ANGPTL4 protein levels were markedly increased at 8 and 16 h following LPS treatment. Administration of zymosan, an activator of TLR2 and a model of fungal infections, also increased serum ANGPTL4 protein and mRNA levels in liver, heart, muscle, and adipose tissue. Finally, treatment of 3T3-L1 adipocytes with LPS or cytokines (TNF alpha, IL-1 beta, and interferon gamma) stimulated ANGPTL4 expression. These studies demonstrate that ANGPTL4 is a positive acute phase protein and the increase in ANGPTL4 could contribute to the hypertriglyceridemia that characteristically occurs during the acute phase response by inhibiting LPL activity.     

Angiopoietin-like Protein 3 Inhibits Lipoprotein Lipase Activity through Enhancing Its Cleavage by Proprotein Convertases

Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.     

Oxfenicine diverts rat muscle metabolism from fatty acid to carbohydrate oxidation and protects the ischaemic rat heart

In isolated diaphragms from rats fed on a high-fat diet, oxfenicine (S-4-hydroxyphenylglycine) stimulated the depressed rates of pyruvate decarboxylation (2-fold) and glucose oxidation (5-fold). In diaphragms from normal-fed rats, oxfenicine had no effect on pyruvate decarboxylation but doubled the rate of glucose oxidation and inhibited the oxidation of palmitate. Treatment of fat-fed rats with oxfenicine restored the proportion of myocardial pyruvate dehydrogenase in the active form to that observed in normal-fed rats. In rat hearts perfused in the presence of glucose, insulin and palmitate, oxfenicine increased carbohydrate oxidation and stimulated cardiac performance with no increase in oxygen consumption - i.e. improved myocardial efficiency. Working rat hearts perfused with glucose, insulin and palmitate and subjected to 10 min global ischaemia recovered to 81% of their pre-ischaemic cardiac output after 30 min reperfusion, and released large amounts of lactate dehydrogenase into the perfusate. Hearts perfused with oxfenicine had slightly higher pre-ischaemic cardiac outputs and, on reperfusion, recovered more completely (to 96% of the pre-ischaemic value). Oxfenicine reduced the amount of lactate dehydrogenase released by 73%. We conclude that, in rat hearts with high rates of fatty acid oxidation, a relative increase in carbohydrate oxidation will improve myocardial efficiency, and preserve mechanical function and cellular integrity during acute ischaemia.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Genetic Variation in ANGPTL4 Provides Insights into Protein Processing and Function

Angiopoietin-like protein 4 (ANGPTL4) is a secreted protein that modulates the disposition of circulating triglycerides (TG) by inhibiting lipoprotein lipase (LPL). Here we examine the steps involved in the synthesis and post-translational processing of ANGPTL4, and the effects of a naturally occurring sequence variant (E40K) that is associated with lower plasma TG levels in humans. Expression of the wild-type and mutant proteins in HEK-293A cells indicated that ANGPTL4 formed dimers and tetramers in cells prior to secretion and cleavage of the protein. After cleavage at a canonical proprotein convertase cleavage site (¹⁶¹RRKR¹⁶⁴), the oligomeric structure of the N-terminal domain was retained whereas the C-terminal fibrinogen-like domain dissociated into monomers. Inhibition of cleavage did not interfere with oligomerization of ANGPTL4 or with its ability to inhibit LPL, whereas mutations that prevented oligomerization severely compromised the capacity of the protein to inhibit LPL. ANGPTL4 containing the E40K substitution was synthesized and processed normally, but no monomers or oligomers of the N-terminal fragments accumulated in the medium; medium from these cells failed to inhibit LPL activity. Parallel experiments performed in mice recapitulated these results. Our findings indicate that oligomerization, but not cleavage, of ANGPTL4 is required for LPL inhibition, and that the E40K substitution destabilizes the protein after secretion, preventing the extracellular accumulation of oligomers and abolishing the ability of the protein to inhibit LPL activity.Angiopoietin-like protein 4 (ANGPTL4)⁴ is a 50-kDa protein that is synthesized and secreted from several metabolically active tissues and has been implicated in the trafficking of circulating TG (1, 2). Triglycerides, either acquired from the diet or synthesized endogenously, circulate in blood as constituents of chylomicrons and very low density lipoproteins (VLDL). As these lipoproteins circulate in tissues they encounter lipoprotein lipase (LPL) at the vascular endothelial surfaces. LPL hydrolyzes the TG, producing free fatty acids that are taken up by the surrounding tissues. ANGPTL4 inhibits the activity of LPL, thereby limiting the uptake of TG-derived fatty acids by the underlying cells (3, 4). Overexpression of ANGPTL4 in mice causes severe hypertriglyceridemia, whereas mice lacking ANGPTL4 have increased LPL activity and low plasma levels of TG (5, 6). In mice, ANGPTL4 is predominantly expressed in adipose tissue and is strongly induced by fasting (2). Accordingly it has been proposed that ANGPTL4 inhibits LPL activity in adipose tissue to reroute fatty acids away from fat to muscle and other tissues when food intake is low (3, 4).ANGPTL4 belongs to a family of seven structurally similar secreted proteins (ANGPTL1-ANGPTL7) that contain a signal sequence followed by an α-helical region predicted to form a coiled-coil, and a globular fibrinogen-like domain at the C terminus (1). Gel filtration studies of recombinant ANGPTL4 indicate that the protein assembles into oligomers that are stabilized by disulfide bonds (7). Substitution of two highly conserved cysteine residues at positions 76 and 80 in the α-helical domain prevents oligomerization of ANGPTL4 and impairs the ability of the recombinant protein to increase plasma TG levels when overexpressed in the livers of rats (7).Upon secretion into the circulation, ANGPTL4 is cleaved into an N-terminal domain and a C-terminal fibrinogen-like domain (8). The N-terminal peptide circulates as an oligomer, and the fibrinogen-like domain circulates as a monomer (8). The N-terminal helical region of ANGPTL4 is necessary and sufficient for inhibition of LPL (9). A peptide corresponding to amino acids 1-187 of the protein binds LPL with high affinity and converts the enzyme from catalytically active dimers to inactive monomers, thereby inhibiting LPL activity (10). After disrupting the LPL dimer, ANGPTL4 is released. The LPL monomers remain folded and stable but fail to re-form active dimers. These data suggest that the N-terminal domain of ANGPTL4 interacts directly but transiently with LPL, triggering a stable conformational switch in LPL that irreversibly inactivates the enzyme.Recently, we used a population-based resequencing strategy to examine the metabolic role of ANGPTL4 in humans (11). Resequencing the coding region of ANGPTL4 in a large (n = 3,501), multiethnic sample revealed multiple rare sequence variations that alter an amino acid in the protein and are associated with low plasma TG levels. In addition, we identified a more common variant (E40K), that was present in ∼3% of European-Americans and was associated with significantly lower plasma levels of TG and low density lipoprotein-cholesterol (LDL-C), and higher levels of high density lipoprotein (HDL)-C in two large epidemiological studies (11). These association studies confirmed that ANGPTL4 is involved in TG metabolism in humans, and also revealed additional roles in humans in the metabolism of HDL and LDL, which were not apparent from studies in genetically modified mice.Here we examined the synthesis, secretion, and processing of ANGPTL4 and determine the mechanism by which substitution of a basic (lysine) for an acidic (glutamate) residue at residue 40 affects the function of the protein.     

Angiopoietin like protein 4 expression is decreased in activated macrophages

Angiopoietin like protein 4 (ANGPTL4) inhibits lipoprotein lipase (LPL) activity. Previous studies have shown that Toll-like Receptor (TLR) activation increases serum levels of ANGPTL4 and expression of ANGPTL4 in liver, heart, muscle, and adipose tissue in mice. ANGPTL4 is expressed in macrophages and is induced by inflammatory saturated fatty acids. The absence of ANGPTL4 leads to the increased uptake of pro-inflammatory saturated fatty acids by macrophages in the mesentery lymph nodes due to the failure of ANGPTL4 to inhibit LPL activity, resulting in peritonitis, intestinal fibrosis, weight loss, and death. Here we determined the effect of TLR activation on the expression of macrophage ANGPTL4. LPS treatment resulted in a 70% decrease in ANGPTL4 expression in mouse spleen, a tissue enriched in macrophages. In mouse peritoneal macrophages, LPS treatment also markedly decreased ANGPTL4 expression. In RAW cells, a macrophage cell line, LPS, zymosan, poly I:C, and imiquimod all inhibited ANGPTL4 expression. In contrast, neither TNF, IL-1, nor IL-6 altered ANGPTL4 expression. Finally, in cholesterol loaded macrophages, LPS treatment still decreased ANGPTL4 expression. Thus, while in most tissues ANGPTL4 expression is stimulated by inflammatory stimuli, in macrophages TLR activators inhibit ANGPTL4 expression, which could lead to a variety of down-stream effects important in host defense and wound repair.     

Inactivation of lipoprotein lipase occurs on the surface of THP-1 macrophages where oligomers of angiopoietin-like protein 4 are formed

Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins causing release of fatty acids for metabolic purposes in muscles and adipose tissue. LPL in macrophages in the artery wall may, however, promote foam cell formation and atherosclerosis. Angiopoietin-like protein (ANGPTL) 4 inactivates LPL and ANGPTL4 expression is controlled by peroxisome proliferator-activated receptors (PPAR). The mechanisms for inactivation of LPL by ANGPTL4 was studied in THP-1 macrophages where active LPL is associated with cell surfaces in a heparin-releasable form, while LPL in the culture medium is mostly inactive. The PPARδ agonist GW501516 had no effect on LPL mRNA, but increased ANGPTL4 mRNA and caused a marked reduction of the heparin-releasable LPL activity concomitantly with accumulation of inactive, monomeric LPL in the medium. Intracellular ANGPTL4 was monomeric, while dimers and tetramers of ANGPTL4 were present in the heparin-releasable fraction and medium. GW501516 caused an increase in the amount of ANGPTL4 oligomers on the cell surface that paralleled the decrease in LPL activity. Actinomycin D blocked the effects of GW501516 on ANGPTL4 oligomer formation and prevented the inactivation of LPL. Antibodies against ANGPTL4 interfered with the inactivation of LPL. We conclude that inactivation of LPL in THP-1 macrophages primarily occurs on the cell surface where oligomers of ANGPTL4 are formed.     

Metabolic response to an acute jump in cardiac workload: effects on malonyl-CoA, mechanical efficiency, and fatty acid oxidation

Inhibition of myocardial fatty acid oxidation can improve left ventricular (LV) mechanical efficiency by increasing LV power for a given rate of myocardial energy expenditure. This phenomenon has not been assessed at high workloads in nonischemic myocardium; therefore, we subjected in vivo pig hearts to a high workload for 5 min and assessed whether blocking mitochondrial fatty acid oxidation with the carnitine palmitoyltransferase-I inhibitor oxfenicine would improve LV mechanical efficiency. In addition, the cardiac content of malonyl-CoA (an endogenous inhibitor of carnitine palmitoyltransferase-I) and activity of acetyl-CoA carboxylase (which synthesizes malonyl-CoA) were assessed. Increased workload was induced by aortic constriction and dobutamine infusion, and LV efficiency was calculated from the LV pressure-volume loop and LV energy expenditure. In untreated pigs, the increase in LV power resulted in a 2.5-fold increase in fatty acid oxidation and cardiac malonyl-CoA content but did not affect the activation state of acetyl-CoA carboxylase. The activation state of the acetyl-CoA carboxylase inhibitory kinase AMP-activated protein kinase decreased by 40% with increased cardiac workload. Pretreatment with oxfenicine inhibited fatty acid oxidation by 75% and had no effect on cardiac energy expenditure but significantly increased LV power and LV efficiency (37 +/- 5% vs. 26 +/- 5%, P < 0.05) at high workload. In conclusion, 1) myocardial fatty acid oxidation increases with a short-term increase in cardiac workload, despite an increase in malonyl-CoA concentration, and 2) inhibition of fatty acid oxidation improves LV mechanical efficiency by increasing LV power without affecting cardiac energy expenditure.     

Angiopoietin-like Protein 4 Inhibition of Lipoprotein Lipase: EVIDENCE FOR REVERSIBLE COMPLEX FORMATION*

Elevated triglycerides are associated with an increased risk of cardiovascular disease, and lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides from circulating lipoproteins. The N-terminal domain of angiopoietin-like protein 4 (ANGPTL4) inhibits LPL activity. ANGPTL4 was previously described as an unfolding molecular chaperone of LPL that catalytically converts active LPL dimers into inactive monomers. Our studies show that ANGPTL4 is more accurately described as a reversible, noncompetitive inhibitor of LPL. We find that inhibited LPL is in a complex with ANGPTL4, and upon dissociation, LPL regains lipase activity. Furthermore, we have generated a variant of ANGPTL4 that is dependent on divalent cations for its ability to inhibit LPL. We show that LPL inactivation by this regulatable variant of ANGPTL4 is fully reversible after treatment with a chelator.     

Reduced cGMP signaling activates NF-kappaB in hypertrophied hearts of mice lacking natriuretic peptide receptor-A

Mice lacking natriuretic peptide receptor-A (NPRA) develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for cardiac hypertrophic growth in the absence of NPRA signaling are not yet known. We sought to determine the activation of nuclear factor-kappaB (NF-kappaB) in Npr1 (coding for NPRA) gene-knockout (Npr1-/-) mice exhibiting cardiac hypertrophy and fibrosis. NF-kappaB binding activity was 4-fold greater in the nuclear extract of Npr1-/- mutant mice hearts as compared with wild-type (Npr1+/+) mice hearts. In parallel, inhibitory kappaB kinase-beta activity and IkappaB-alpha protein phosphorylation were also increased 3- and 4-fold, respectively, in hypertrophied hearts of mutant mice. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates NF-kappaB binding activity associated with hypertrophic growth of mutant mice hearts.     

Reduced inhibitor 1 and 2 activity is associated with increased protein phosphatase type 1 activity in left ventricular myocardium of one-kidney, one-clip hypertensive rats

In failing hearts, although protein phosphatase type 1 (PP1) activity has increased, information about the regulation and status of PP1 inhibitor-1 (INH-1) and inhibitor-2 (INH-2) is limited. In this study, we examined activity and protein expression of PP1, INH-1 and INH-2 and phosphorylation of sarcoplasmic reticulum (SR) phospholamban (PLB), a substrate of PP1 and modulator of SR Ca2+-ATPase activity, in failing and non-failing hearts. These studies were performed in LV myocardium of seven rats with chronic renal hypertension produced by Goldblatt's one-kidney, one-clip procedure and seven age-matched sham-operated normal controls (CTR). Eight weeks after surgery, LV ejection fraction, LV hypertrophy, and pulmonary congestion were determined in all rats. PP1 activity (nmol 32P/min/mg non-collagen protein) was assessed in LV homogenates using 32P-labeled phosphorylase a as substrate. INH-1 and INH-2 activity was determined in the immunoprecipitate of LV homogenates and expressed as percentage inhibitory activity. Using a specific antibody, LV tissue levels of PP1C and calsequestrin (CSQ), a SR calcium binding protein, which is not altered in failing hearts, were also determined. Further, total and phosphorylated PLB, INH-1 and INH-2 protein levels were determined in the LV homogenate and phosphoprotein-enriched fraction, respectively. The band density of each protein was quantified in densitometric units and normalized to CSQ. Results: rats with chronic renal hypertension exhibited significantly reduced LV ejection fraction and increased LV hypertrophy and pulmonary congestion, characteristics of chronic heart failure (CHF). We found that compared to CTR, (1) both INH-1 (10.2+/-2 versus 57.5+/-1; p < 0.05) and INH-2 activity (3.8+/-0.4 versus 36.2+/-4; p < 0.05) were reduced, (2) total and phosphorylated PLB amount reduced, (3) protein level of phosphorylated INH-1 was reduced (2.32+/-0.1 versus 0.73+/-0.04; p < 0.05) whereas that of phosphorylated INH-2 increased (3.05+/-0.3 versus 1.42+/-0.1; p < 0.05), and (4) PP1 activity was increased approximately 2.6-fold in rats with CHF (1.59+/-0.05 versus 0.61+/-0.01; p < 0.05) while protein level of the catalytic subunit of PP1 (PP1C) increased 3.85-fold (0.77+/-0.05 versus 0.20+/-0.02; p < 0.05). These results suggest that reduced inhibitory INH-1 and INH-2 activity, increased PP1C protein level, and reduced PLB phosphorylation are associated with increased PP1 activity in failing hearts.     

Lipoprotein lipase deficiency is associated with elevated acylation stimulating protein plasma levels

Acylation stimulating protein (ASP, C3adesArg) is an adipose tissue derived hormone that stimulates triglyceride (TG) synthesis. ASP stimulates lipoprotein lipase (LPL) activity by relieving feedback inhibition caused by fatty acids (FA). The present study examines plasma ASP and lipids in male and female LPL-deficient subjects primarily with the P207L mutation, common in the population of Quebec, Canada. We evaluated the fasting and postprandial states of LPL heterozygotes and fasting levels in LPL homozygotes. Homozygotes displayed increased ASP (58–175% increase, P < 0.05–0.01), reduced HDL-cholesterol (64–75% decrease, P < 0.0001), and elevated levels of TG (19–38-fold, P < 0.0001) versus control (CTL) subjects. LPL heterozygotes with normal fasting TG (1.3–1.9 mmol/l) displayed increased ASP (101–137% increase, P < 0.05–0.01) and delayed TG clearance after a fatload; glucose levels remained similar to controls. Hypertriglyceridemics with no known LPL mutation also had increased ASP levels (63–192% increase, P < 0.001). High-TG LPL heterozygotes were administered a fatload before and after fibrate treatment. The treatment reduced fasting and postprandial plasma ASP, TG, and FA levels without changing insulin or glucose levels. ASP enhances adipose tissue fatty-acid trapping following a meal; however in LPL deficiency, high ASP levels are coupled with delayed lipid clearance.     

Proteolytic processing of angiopoietin-like protein 4 by proprotein convertases modulates its inhibitory effects on lipoprotein lipase activity

Angiopoietin-like protein 4 (ANGPTL4) has been associated with a variety of diseases. It is known as an endogenous inhibitor of lipoprotein lipase (LPL), and it modulates lipid deposition and energy homeostasis. ANGPTL4 is cleaved by unidentified protease(s), and the biological importance of this cleavage event is not fully understood with respect to its inhibitory effect on LPL activity. Here, we show that ANGPTL4 appears on the cell surface as the full-length form, where it can be released by heparin treatment in culture and in vivo. ANGPTL4 protein is then proteolytically cleaved into several forms by proprotein convertases (PCs). Several PCs, including furin, PC5/6, paired basic amino acid-cleaving enzyme 4, and PC7, are able to cleave human ANGPTL4 at a consensus site. PC-specific inhibitors block the processing of ANGPTL4. Blockage of ANGPTL4 cleavage reduces its inhibitory effects on LPL activity and decreases its ability to raise plasma triglyceride levels. In summary, the cleavage of ANGPTL4 by these PCs modulates its inhibitory effect on LPL activity.     

ANGPTL3 blockade with a human monoclonal antibody reduces plasma lipids in dyslipidemic mice and monkeys

Angiopoietin-like protein 3 (ANGPTL3) is a circulating protein synthesized exclusively in the liver that inhibits LPL and endothelial lipase (EL), enzymes that hydrolyze TGs and phospholipids in plasma lipoproteins. Here we describe the development and testing of a fully human monoclonal antibody (REGN1500) that binds ANGPTL3 with high affinity. REGN1500 reversed ANGPTL3-induced inhibition of LPL activity in vitro. Intravenous administration of REGN1500 to normolipidemic C57Bl/6 mice increased LPL activity and decreased plasma TG levels by ≥50%. Chronic administration of REGN1500 to dyslipidemic C57Bl/6 mice for 8 weeks reduced circulating plasma levels of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) without any changes in liver, adipose, or heart TG contents. Studies in EL knockout mice revealed that REGN1500 reduced serum HDL-C through an EL-dependent mechanism. Finally, administration of a single dose of REGN1500 to dyslipidemic cynomolgus monkeys caused a rapid and pronounced decrease in plasma TG, nonHDL-C, and HDL-C. REGN1500 normalized plasma TG levels even in monkeys with a baseline plasma TG greater than 400 mg/dl. Collectively, these data demonstrate that neutralization of ANGPTL3 using REGN1500 reduces plasma lipids in dyslipidemic mice and monkeys, and thus provides a potential therapeutic agent for treatment of patients with hyperlipidemia.     

Omega-3 long-chain fatty acids strongly induce angiopoietin-like 4 in humans

Angiopoietin-like 4 (ANGPTL4) is a regulator of LPL activity. In this study we examined whether different fatty acids have a differential effect on plasma ANGPTL4 levels during hyperinsulinemia in healthy lean males. In 10 healthy lean males, 3 hyperinsulinemic euglycemic clamps were performed during concomitant 6 h intravenous infusion of soybean oil (Intralipid® rich in PUFA), olive oil (Clinoleic® rich in MUFA) and control saline. In 10 other healthy lean males, 2 hyperinsulinemic clamps were performed during infusion of a mixed lipid emulsion containing a mixture of fish oil (FO), medium-chain triglycerides (MCTs), and long-chain triglycerides (LCTs) (FO/MCT/LCT; SMOFlipid®) or saline. FFA levels of approximately 0.5 mmol/l were reached during each lipid infusion. Plasma ANGPTL4 decreased during hyperinsulinemia by 32% (18–52%) from baseline. This insulin-mediated decrease in ANGPTL4 concentrations was partially reduced during concomitant infusion of olive oil and completely blunted during concomitant infusion of soybean oil and FO/MCT/LCT. The reduction in insulin sensitivity was similar between all lipid infusions. In accordance, incubation of rat hepatoma cells with the polyunsaturated fatty acid C22:6 increased ANGPTL4 expression by 70-fold, compared with 27-fold by the polyunsaturated fatty acid C18:2, and 15-fold by the monounsaturated fatty acid C18:1. These results suggest that ANGPTL4 is strongly regulated by fatty acids in humans, and is also dependent on the type of fatty acid.