Inhibition Of Lipoprotein Lipase Activity Research Articles

Disruption of the PGE2 synthesis / response pathway restrains atherogenesis in programmed cell death-1 (Pd-1) deficient hyperlipidemic mice.

Immune checkpoint inhibitors (ICIs) that target programmed cell death 1 (PD-1) have revolutionized cancer treatment by enabling the restoration of suppressed T-cell cytotoxic responses. However, resistance to single-agent ICIs limits their clinical utility. Combinatorial strategies enhance their antitumor effects, but may also enhance the risk of immune related adverse effects of ICIs. Prostaglandin (PG) E2, formed by the sequential action of the cyclooxygenase (COX) and microsomal PGE synthase (mPGES-1) enzymes, acting via its E prostanoid (EP) receptors, EPr2 and EPr4, promotes lymphocyte exhaustion, revealing an additional target for ICIs. Thus, COX inhibitors and EPr4 antagonists are currently being combined with ICIs potentially to enhance antitumor efficacy in clinical trials. However, given the cardiovascular (CV) toxicity of COX inhibitors, such combinations may increase the risk particularly of CV AEs. Here, we compared the impact of distinct approaches to disruption of the PGE2 synthesis /response pathway - global or myeloid cell specific depletion of mPges-1 or global depletion of Epr4 - on the accelerated atherogenesis in Pd-1 deficient hyperlipidemic (Ldlr-/-) mice. All strategies restrained the atherogenesis. While depletion of mPGES-1 suppresses PGE2 biosynthesis, reflected by its major urinary metabolite, PGE2 biosynthesis was increased in mice lacking EPr4, consistent with enhanced expression of aortic Cox-1 and mPges-1. Deletions of mPges-1 and Epr4 differed in their effects on immune cell populations in atherosclerotic plaques; the former reduced neutrophil infiltration, while the latter restrained macrophages and increased the infiltration of T-cells. Consistent with these findings, chemotaxis by bone-marrow derived macrophages from Epr4-/- mice was impaired. Epr4 depletion also resulted in extramedullary lymphoid hematopoiesis and inhibition of lipoprotein lipase activity (LPL) with coincident spelenomegaly, leukocytosis and dyslipidemia. Targeting either mPGES-1 or EPr4 may restrain lymphocyte exhaustion while mitigating CV irAEs consequent to PD-1 blockade.

ANGPTL3 accelerates atherosclerotic progression via direct regulation of M1 macrophage activation in plaque

IntroductionThe N-terminal domain of angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein lipase activity. Its C-terminal fibrinogen-like (FBN) domain is a ligand of macrophage integrin αvβ3. ObjectivesANGPTL3 might home to plaque where it directly regulates macrophage function via integrin αvβ3 for atherosclerosis progression. MethodsLdlr-/- mice on a high-fat diet and ApoE-/- mice on a chow diet were received adeno-associated virus (AAV)-mediated Angptl3 gene transfer and followed up for 12 weeks. ApoE-/- mice were injected AAV containing FLAG-tagged Angptl3 cDNA for tracing. Atherosclerotic features were compared between Angptl3-/-ApoE-/- mice and ApoE-/- littermates. THP-1 cells were exposed to 0 or 50 μg/ml ANGPTL3 FBN domain for 24 h to evaluate Toll-like receptor (TLR)4 expression using western blot analysis and circulating cytokine and chemokine profiles by the MILLIPLEX MAP assay. Phospho-proteomic profile was established in ANGPTL3-treated macrophages. Integrin β3 deficient THP-1 cells were obtained by sgRNAs targeting RGD sequence using Lentivirus-Cas9 system. ResultsAngptl3 overexpression increased atherosclerotic progression and CD68+ macrophages in plaque (p < 0.05 for all). By immunostaining, FLAG+ cells were identified in plaque of gene transferred ApoE-/- mice. Fluorescent immunostaining detected co-localisation of Angptl3 and CD68 in plaque macrophages. Phospho-proteomic analysis revealed that Angptl3 induced phosphorylation of proteins that were involved in the IL-17 signalling pathway in THP-1 cells. In vitro, ANGPTL3 treatment increased the production of interleukin (IL)-1β and tumour necrosis factor-α in THP-1 cells (p < 0.05 for both). Exposure of ANGPTL3 to THP-1 cells induced Akt phosphorylation which was weakened in integrin β3 deficient ones. ANGPTL3 elevated TLR4 expression via Akt phosphorylation. In response to lipopolysaccharide, nuclear factor-κB activity was 2.2-fold higher in THP-1 cells pre-treated with ANGPTL3 than in untreated cells (p < 0.05). ConclusionsTargeting ANGPTL3 could yield a dual benefit of lowering lipid levels in the blood and suppressing macrophage activation in plaque.

Friend or foe? The elusive role of ANGPTL4 in inflammation

Angiopoietin-like 4 (ANGPTL4) belongs to the angiopoietin-like protein family and mediates the inhibition of lipoprotein lipase activity. Emerging evidence suggests that ANGPTL4 has pleiotropic functions with anti- and pro-inflammatory properties. Here, we have reviewed the research progress on ANGPTL4 and systematically discussed the dual role of ANGPTL4 in inflammation and inflammatory diseases. Understanding the potential mechanisms of ANGPTL4 in inflammation will aid in drug discovery and treatment development.

ANGPTL4 May Regulate the Crosstalk Between Intervertebral Disc Degeneration and Type 2 Diabetes Mellitus: A Combined Analysis of Bioinformatics and Rat Models.

The crosstalk between intervertebral disc degeneration (IVDD) and type 2 diabetes mellitus (T2DM) has been investigated. However, the common mechanism underlying this phenomenon has not been clearly elucidated. This study aimed to explore the shared gene signatures of IVDD and T2DM. The expression profiles of IVDD (GSE27494) and T2DM (GSE20966) were acquired from the Gene Expression Omnibus database. Five hub genes including ANGPTL4, CCL2, CCN3, THBS2, and INHBA were preliminarily screened. GO (Gene Ontology) enrichment analysis, functional correlation analysis, immune filtration, Transcription factors (TFs)-mRNA-miRNA coregulatory network, and potential drugs prediction were performed following the identification of hub genes. RNA sequencing, in vivo and in vitro experiments on rats were further performed to validate the expression and function of the target gene. Five hub genes (ANGPTL4, CCL2, CCN3, THBS2, and INHBA) were identified. GO analysis demonstrated the regulation of the immune system, extracellular matrix (ECM), and SMAD protein signal transduction. There was a strong correlation between hub genes and different functions, including lipid metabolism, mitochondrial function, and ECM degradation. The immune filtration pattern grouped by disease and the expression of hub genes showed significant changes in the immune cell composition. TFs-mRNA-miRNA co-expression networks were constructed. In addition, pepstatin showed great drug-targeting relevance based on potential drugs prediction of hub genes. ANGPTL4, a gene that mediates the inhibition of lipoprotein lipase activity, was eventually determined after hub gene screening, validation by different datasets, RNA sequencing, and experiments. This study screened five hub genes and ANGPTL4 was eventually determined as a potential target for the regulation of the crosstalk in patients with IVDD and T2DM.

Targeted Metabolomics Analysis of Individuals Carrying the ANGPTL8 R59W Variant.

ANGPTL8 is recognized as a regulator of lipid metabolism through its role in inhibiting lipoprotein lipase activity. ANGPTL8 gene variants, particularly rs2278426 leading to the R59W variant in the protein, have been associated with lipid traits in various ethnicities. We aimed to use metabolomics to understand the impact of the ANGPTL8 R59W variant on metabolites in humans. We used the Biocrates-p400 kit to quantify 408 plasma metabolites in 60 adult male Arab individuals from Kuwait and identify differences in metabolite levels between individuals carrying reference genotypes and those with carrier genotypes at ANGPTL8 rs2278426. Individuals with carrier genotypes (CT+TT) compared to those carrying the reference genotype (CC) showed statistically significant differences in the following metabolites: acylcarnitine (perturbs metabolic pathways), phosphatidylcholine (supports liver function and cholesterol levels), cholesteryl ester (brings chronic inflammatory response to lipoprotein depositions in arteries), α-aminoadipic acid (modulates glucose homeostasis), histamine (regulates glucose/lipid metabolism), sarcosine (links amino acid and lipid metabolism), diacylglycerol 42:1 (regulates homeostasis of cellular lipid stores), and lysophosphatidylcholine (regulates oxidative stress and inflammatory response). Functional aspects attributed to these metabolites indicate that the ANGPTL8 R59W variant influences the concentrations of lipid- and inflammation-related metabolites. This observation further highlights the role of ANGPTL8 in lipid metabolism.

Dual role of ANGPTL4 in inflammation.

Angiopoietin-like 4 (ANGPTL4) belongs to the angiopoietin-like protein family and mediates the inhibition of lipoprotein lipase activity. Emerging evidence suggests that ANGPTL4 has pleiotropic functions with anti- and pro-inflammatory properties. A thorough search on PubMed related to ANGPTL4 and inflammation was performed. Genetic inactivation of ANGPTL4 can significantly reduce the risk of developing coronary artery disease and diabetes. However, antibodies against ANGPTL4 result in several undesirable effects in mice or monkeys, such as lymphadenopathy and ascites. Based on the research progress on ANGPTL4, we systematically discussed the dual role of ANGPTL4 in inflammation and inflammatory diseases (lung injury, pancreatitis, heart diseases, gastrointestinal diseases, skin diseases, metabolism, periodontitis, and osteolytic diseases). This may be attributed to several factors, including post-translational modification, cleavage and oligomerization, and subcellular localization. Understanding the potential underlying mechanisms of ANGPTL4 in inflammation in different tissues and diseases will aid in drug discovery and treatment development.

Molecular characterization and immune functions of lipasin in Nile tilapia (Oreochromis niloticus): Involvement in the regulation of tumor necrosis factor-α secretion

Lipasin, the product of the angiopoietin-like 8 (angptl8) gene, is known as a critical regulator of plasma lipid metabolism. However, its immune function in vertebrates is currently poorly understood. By 5'/3′-rapid amplification of cDNA ends (RACE), we established the structural identity of Nile tilapia (Oreochromis niloticus) angptl8. The transcripts of tilapia angptl8 were widely expressed in various tissues, with the highest levels in the liver. Following lipopolysaccharide in vivo challenges, time-dependent angptl8 gene expression was observed in the head kidney and liver. On the basis of the sequence obtained, we produced recombinant lipasin that inhibited lipoprotein lipase activity. Treatment of head kidney leukocytes with lipasin stimulated tumor necrosis factor-α (TNF-α) secretion and gene expression. In addition, lipasin-induced TNF-α secretion could be prevented by inhibiting the nuclear factor-kappa B (NF-κB) signaling pathway. Furthermore, lipasin enhanced the phosphorylation and degradation of IκBα and promoted translocation of the p65 subunit of NF-κB to the nucleus. Collectively, the current findings suggested that lipasin was involved in the immune response of Nile tilapia and stimulated TNF-α secretion by activating the NF-κB pathway in tilapia head kidney leukocytes.

Evinacumab for the treatment of homozygous familial hypercholesterolemia

ABSTRACT Introduction Hypercholesterolemia is mainly caused by abnormal lipoprotein metabolism and can increase the risk of cardiovascular disease. Angiopoietin-like protein 3 (ANGPTL3) can increase low-density lipoprotein cholesterol (LDL-C) and other lipids by inhibiting lipoprotein lipase activity. Evinacumab is a monoclonal antibody against ANGPTL3, it can decrease levels of LDL-C and has shown potential benefit in patients with homozygous familial hypercholesterolemia (HoFH). Areas covered A comprehensive literature search was conducted in PubMed (January 2000 to August 2021). Key search terms included ANGPTL3, evinacumab and HoFH. Other sources were derived from product labeling and ClinicalTrials.gov. All English-language articles identified from the data sources were reviewed and evaluated. Phase 1, 2 and 3 clinical trials were included. The pharmacological characteristics, clinical evidence, and safety of evinacumab were reviewed. Expert opinion Evinacumab is an ANGPTL3 inhibitor. Phase 3 clinical trials found that in patients with HoFH, evinacumab reduced LDL-C by 47%, but placebo increased by 2%. Evinacumab was well-tolerated. Common adverse events included nasopharyngitis, influenza-like illness, dizziness, rhinorrhea, and nausea. It has the potential to become a valuable treatment option for patients with HoFH.

ANGPTL8 in metabolic homeostasis: more friend than foe?

ANGPTL8 is an important cytokine, which is significantly increased in type 2 diabetes mellitus (T2DM), obesity and metabolic syndrome. Many studies have shown that ANGPTL8 can be used as a bio-marker of these metabolic disorders related diseases, and the baseline ANGPTL8 level has also been found to be positively correlated with retinopathy and all-cause mortality in patients with T2DM. This may be related to the inhibition of lipoprotein lipase activity and the reduction of circulating triglyceride (TG) clearance by ANGPTL8. Consistently, inhibition of ANGPTL8 seems to prevent or improve atherosclerosis. However, it is puzzling that ANGPTL8 seems to have a directing function for TG uptake in peripheral tissues; that is, ANGPTL8 specifically enhances the reserve and buffering function of white adipose tissue, which may alleviate the ectopic lipid accumulation to a certain extent. Furthermore, ANGPTL8 can improve insulin sensitivity and inhibit hepatic glucose production. These contradictory results lead to different opinions on the role of ANGPTL8 in metabolic disorders. In this paper, the correlation between ANGPTL8 and metabolic diseases, the regulation of ANGPTL8 and the physiological role of ANGPTL8 in the process of glucose and lipid metabolism were summarized, and the physiological/pathological significance of ANGPTL8 in the process of metabolic disorder was discussed.

New Classification and Management of Abetalipoproteinemia and Related Disorders

New Classification and Management of Abetalipoproteinemia and Related Disorders

Intricacies of the endothelin system in human obesity: role in the development of complications and potential as a therapeutic target.

Activation of the vascular endothelin-1 (ET-1) system is a key abnormality in vascular dysfunction of human obesity, especially in patients developing complications, such as the metabolic syndrome, diabetes, and atherosclerosis. Vascular insulin resistance, an increased insulin-stimulated endothelial production of ET-1 combined with impaired nitric oxide availability, is the hallmark of obesity-related vasculopathy, but dysregulated adipokine release from obese adipose tissue may contribute to the predominance of ET-1-dependent vasoconstriction. ET-1, in turn, might determine unhealthy obese adipose tissue expansion, with visceral and perivascular adipose tissue changes driving the release of inflammatory cytokines and atherogenic chemokines. In addition, ET-1 might also play a role in the development of the metabolic complications of obesity. Studies have shown inhibition of lipoprotein lipase activity by ET-1, with consequent hypertriglyceridemia. Also, ET-1 in pancreatic islets seems to contribute to beta cell dysfunction, hence affecting insulin production and development of diabetes. Moreover, ET-1 may play a role in nonalcoholic steatohepatitis. Recent clinical trials using innovative design have demonstrated that antagonism of ET-type A receptors protects against some complications of obesity and diabetes, such as nephropathy. These findings encourage further investigation to evaluate whether targeting the ET-1 system could afford better protection against other consequences of the obesity epidemic.

Induction of Free Fatty Acid‐mediated Angiopoietin‐Like Protein‐4 Gene Expression is Independent of PPARδ Activity in Skeletal Muscle Cells In Vitro

Angiopoietin‐like protein‐4 (ANGPTL4) inhibits lipoprotein lipase activity in skeletal muscle. Free fatty acids (FFAs) activate ANGPTL4 expression, yet the method by which FFAs induce ANGPTL4 in skeletal muscle is not well‐known. The purpose of this study was to examine the effect of FFAs on peroxisome proliferator‐activated receptor δ (PPARδ)‐mediated ANGPTL4 expression in C2C12 cells. Chronic exposure of mono‐ (MUFA) and poly‐unsaturated fatty acids (PUFA), but not saturated fatty acids (SFA), significantly induced ANGPTL4 mRNA expression in C2C12 cells (MUFA: 4.5±1.1, p=0.02; PUFA: 5.6±1.6, p=0.02; SFA: 0.4±0.1, p=0.4 respectively). To analyze the mechanism of FFA‐induced PPARδ activation of ANGPTL4, mRNA expression of PPARδ, which is believed to induce ANGPTL4 activity, was measured. However, there was no significant difference in PPARδ expression after FFA treatments. Next, we used the PPARδ agonist (GW0742) and antagonist (GSK0660) to modulate PPARδ activity independent of FFA treatment and examined ANGPTL4 expression. GW0742 significantly increased ANGPTL4 in undifferentiated and differentiated C2C12 cells (12.6±3.9, p=0.01 and 2.8±0.2, p&lt;0.001 respectively), while GSK0660 significantly suppressed basal ANGPTL4 expression (0.2±0.1, p=0.05 and 0.2±0.03, p&lt;0.001 respectively). Yet, in the presence of FFA and GSK0660, ANGPTL4 gene expression significantly increased despite the absence of PPARδ activity in both undifferentiated and differentiated C2C12 cells (7.1±2.5, p=0.03 and 3.8±0.4, p&lt;0.001). Taken together, these results indicate that FFAs may activate ANGPTL4 in a PPARδ independent manner, and a revised mechanism of FFA‐induced skeletal muscle ANGPTL4 expression may be warranted.

APOC3-rs2854116 polymorphism related to hypercholesterolemia in primary school children in Hanoi

APOC3-rs2854116 polymorphism related to hypercholesterolemia in primary school children in Hanoi

Effect of mountain ultra-marathon running on plasma angiopoietin-like protein 4 and lipid profile in healthy trained men

PurposeAngiopoietin-like protein 4 (ANGPTL4) regulates lipid metabolism by inhibiting lipoprotein lipase activity and stimulating lipolysis in adipose tissue. The aim of this study was to find out whether the mountain ultra-marathon running influences plasma ANGPTL4 and whether it is related to plasma lipid changes.MethodsTen healthy men (age 31 ± 1.1 years) completed a 100-km ultra-marathon running. Plasma ANGPTL4, free fatty acids (FFA), triacylglycerols (TG), glycerol (Gly), total cholesterol (TC), low (LDL-C) and high (HDL-C) density lipoprotein-cholesterol were determined before, immediately after the run and after 90 min of recovery.ResultsPlasma ANGPTL4 increased during exercise from 68.0 ± 16.5 to 101.2 ± 18.1 ng/ml (p < 0.001). This was accompanied by significant increases in plasma FFA, Gly, HDL-C and decreases in plasma TG concentrations (p < 0.01). After 90 min of recovery, plasma ANGPTL4 and TG did not differ significantly from the exercise values, while plasma FFA, Gly, TC and HDL-C were significantly lower than immediately after the run.TC/HDL-C and TG/HDL-C molar ratios were significantly reduced. The exercise-induced changes in plasma ANGPTL4 correlated positively with those of FFA (r = 0.73; p < 0.02), and HDL-C (r = 0.69; p < 0.05). Positive correlation was found also between plasma ANGPTL4 and FFA concentrations after 90 min of recovery (r = 0.77; p < 0.01).ConclusionsThe present data suggest that increase in plasma FFA during mountain ultra-marathon run may be involved in plasma ANGPTL4 release and that increase in ANGPTL4 secretion may be a compensatory mechanism against fatty acid-induced oxidative stress. Increase in plasma HDL-C observed immediately after the run may be due to the protective effect of ANGPTL4 on HDL.

Apolipoprotein C-II mimetic peptide is an efficient activator of lipoprotein lipase in human plasma as studied by a calorimetric approach

Elevated plasma triglyceride (TG) levels are associated with higher risk of atherosclerotic cardiovascular disease. One way to reduce plasma TG is to increase the activity of lipoprotein lipase (LPL), the rate limiting enzyme in plasma TG metabolism. An apolipoprotein (apo) C-II mimetic peptide (18A–CII–a) has been recently developed that stimulated LPL activity in vitro and decreased plasma TG concentration in animal models for hypertriglyceridemia. Since this peptide can serve as a new therapeutic approach for treatment of hypertriglyceridemia, we investigated how 18A–CII–a peptide influences LPL activity in human plasma. We used recently described isothermal titration calorimetry based approach to assess the peptide, which enables the analysis in nearly undiluted human plasma. The 18A–CII–a peptide was 3.5-fold more efficient in stimulating LPL activity than full-length apoC-II in plasma sample from normolipidemic individual. Furthermore, 18A–CII–a also increased LPL activity in hypertriglyceridemic plasma samples. Unlike apoC-II, high concentrations of the 18A–CII–a peptide did not inhibit LPL activity. The increase in LPL activity after addition of 18A–CII–a or apoC-II to plasma was due to the increase of the amount of available substrate for LPL. Measurements with isolated lipoproteins revealed that the relative activation effects of 18A–CII–a and apoC-II on LPL activity were greater in smaller size lipoprotein fractions, such as remnant lipoproteins, low-density lipoproteins and high-density lipoproteins. In summary, this report describes a novel mechanism of action for stimulation of LPL activity by apoC-II mimetic peptides.

Hypocholesterolemia and dysregulated production of angiopoietin-like proteins in sickle cell anemia patients

Angiopoietin-like proteins (ANGPTL) are responsible for inhibiting lipoprotein lipase activity, and ANGPTL3 and ANGPTL4 deficiencies have been shown to lower lipoprotein levels in animal models and in humans carrying loss-of-function mutations. Sickle cell anemia (SCA) is a hereditary hemolytic anemia characterized by vaso-occlusive crises and end-organ damage, which is curiously associated with hypocholesterolemia and a low incidence of atherosclerosis, whose underlying mechanisms are unclear. We hypothesized that ANGPTL3 and ANGPTL4 dysregulation is responsible for the hypolipidemic phenotype in SCA. We measured circulating concentrations of ANGPTL3 and ANGPTL4 and correlated them with hemolytic biomarkers and lipoproteins in 40 patients with SCA and 30 control individuals. The association between hemolysis and low cholesterol levels in SCA was confirmed along with surprisingly higher levels of ANGPTL3 and ANGPTL4 in SCA patients than in controls. ANGPTL3 correlated with hemolysis markers LDH and reticulocyte counts, while ANGPTL4 did not. Our data show a paradoxical increase in production of ANGPTL3 and ANGPTL4 in SCA, which would be expected to cause hyperlipidemia, due to increased inhibition of lipoprotein lipase. ANGPTL3, exclusively produced by the liver, correlated with hemolysis markers, suggesting a possible hepatic response to hemolysis. Further functional studies and replication in larger cohorts are warranted to investigate the dysregulation of lipid metabolism in SCA.

Lipoprotein lipase in mouse kidney: effects of nutritional status and high-fat diet

Activity of lipoprotein lipase (LPL) is high in mouse kidney, but the reason is poorly understood. The aim was to characterize localization, regulation, and function of LPL in kidney of C57BL/6J mice. We found LPL mainly in proximal tubules, localized inside the tubular epithelial cells, under all conditions studied. In fed mice, some LPL colocalized with the endothelial markers CD31 and GPIHBP1 and could be removed by perfusion with heparin, indicating a vascular location. The role of angiopoietin-like protein 4 (ANGPTL4) for nutritional modulation of LPL activity was studied in wild-type and Angptl4-/- mice. In Angptl4-/- mice, kidney LPL activity remained high in fasted animals, indicating that ANGPTL4 is involved in suppression of LPL activity on fasting, like in adipose tissue. The amount of ANGPTL4 protein in kidney was low, and the protein appeared smaller in size, compared with ANGPTL4 in heart and adipose tissue. To study the influence of obesity, mice were challenged with high-fat diet for 22 wk, and LPL was studied after an overnight fast compared with fasted mice given food for 3 h. High-fat diet caused blunting of the normal adaptation of LPL activity to feeding/fasting in adipose tissue, but in kidneys this adaptation was lost only in male mice. LPL activity increases to high levels in mouse kidney after feeding, but as no difference in uptake of chylomicron triglycerides in kidneys is found between fasted and fed states, our data confirm that LPL appears to have a minor role for lipid uptake in this organ.

Thiazolidinedione induces a therapeutic effect on hepatosteatosis by regulating stearoyl-CoA desaturase-1, lipase activity, leptin and resistin.

Hepatosteatosis is a disease present worldwide, which presents a number of health problems. Recently, thiazolidinedione (TZD) has been used as a therapy for lipid disorders. The present study demonstrates the potential of TZD as a treatment for hepatosteatosis and its mechanism of action, particularly focusing on its role in lipid metabolism. A total of 60 (80–90 g) rats were divided into three groups: A normal group with a standard diet, a high-fat, high-carbohydrate diet (HFCD) group or a HFCD+TZD group (n=20/group). The HFCD induced hepatosteatosis over a period of 12 weeks and the HFCD+TZD group were administered TZD in weeks 13–16. Blood and tissue samples were collected to measure hepatic function, the lipid profile, metabolism and hormone biomarkers, including serum triglyceride (TG), lipoprotein lipase (LPL), stearoyl-CoA desaturase (SCD-1), leptin and resistin. The HFCD-fed rats exhibited a significant increase in serum TG, total cholesterol, low-density lipoproteins, alanine transaminase and bilirubin compared with the normal group as well as a significant decrease in high-density lipoprotein. In addition, serum leptin and resistin were significantly elevated in the HFCD group compared with the normal group. The administration of TZD significantly increased SCD-1 activity and significantly inhibited LPL activity. It also attenuated the changes in the lipid profiles and normalized serum leptin and resistin levels. The results of the present study indicated that HFCD induced lipid abnormalities associated with hypertriglyceridemia, hypercholesterolemia and hepatosteatosis. These changes resulted from disruption to leptin and resistin, which may be due to alterations in LPL and SCD-1 activity. TZD mitigated the effects of HFCD-induced hepatosteatosis, indicating a possible regulatory effect of TZD in the development of hepatosteatosis. The authors suggest that the manipulation of SCD-1 and lipase by TZD may be useful as a treatment for hepatosteatosis.

Abstract 013: Genotype to Phenotype: Function of Rare Coding Variants in ANGPTL3

Angiopoietin-like 3 ( ANGPTL3 ) associates strongly with blood lipid phenotypes in human genetics studies and has thus emerged as promising therapeutic target for plasma lipids. We examined the coding regions of ANGPTL3 and identified 82 rare variants in individuals from separate human cohorts. In light of the seemingly favorable clinical consequences of ANGPTL3 deficiency, we established an experimental framework to determine the functional consequences of rare ANGPTL3 missense mutations in vivo . We generated an Angptl3 knockout mouse, which exhibited decreased triglyceride (TG) (61%, P &lt; 0.001) and decreased total cholesterol (31%, P &lt; 0.002). We attempted to rescue this phenotype using adenoviruses expressing either wild-type or missense variant of ANGPTL3 . To date, all 82 rare missense variants have been assessed, of which 25 were validated in terms of loss-of-function as severe (conferring &lt;25% of wild-type activity as assessed by either TG or cholesterol levels), 18 were moderate (25-50%), 18 were mild (50-75%), and 20 were benign (75-125%) while 1 was gain-of-function (&gt;125%), underscoring the need for functional characterization of variants of uncertain significance. Furthermore, we noticed that 4 variants (H343R, S86L, E129G and T256K) were loss-of-function on triglyceride levels, while benign on cholesterol levels. On the other hand, 7 variants (L75F, C408R, Y350H, K387Q, Q293E, D455E and M259R) were loss-of-function on cholesterol while benign on TG. ANGPTL3 has been implicated in regulating TG and cholesterol through different mechanisms. Therefore, we studied the potential mechanisms by knocking in variants into induced pluripotent stem cells (iPSCs) in vitro . We next differentiated edited cells into hepatocyte-like cells (HLCs). We observed no cleaved N-terminal fragment of ANGPTL3 in HLCs with the E129G variant while the expression level of full-length ANGPTL3 was unaffected. It has been reported that N-terminal fragment is sufficient to inhibit lipoprotein lipase activity which is the primary mechanism by which triglyceride-rich lipoproteins are cleared from the circulation. These results indicate that variant E129G could not be cleaved and this might be the mechanism by which it only affects TG, not cholesterol levels.

Effects of Different Oxygen Concentrations on Human Differentiated Preadipocytes Lipogenic and Lipolytic functions

ContextFat stored in white adipose tissue is the human body's largest energy reservoir. Adipocytes, the predominant cell population in white adipose tissue, play a central role in the clearance of circulating triglyceride (TG) and the delivery of fatty acids for use by peripheral tissues. Impaired regulation of adipose tissue functions is thought to stimulate lipid deposition in non‐adipose tissues, inducing lipotoxic response and the development of metabolic disorders. During weight gain, adipocytes expand in size, decreasing adipose tissue capillary density and creating areas characterized by excessively low oxygen concentrations (hypoxia). It is not clear how hypoxia modulates adipose tissue TG clearance and fatty acid delivery.ObjectiveThe purpose of this study was to investigate the effects of different hypoxia concentrations on lipogenic (store TG) and lipolytic functions (deliver fatty acids) of human differentiated preadipocytes.MethodsDifferentiated human preadipocytes were exposed to different oxygen concentrations (3%, 10%, and 21%) either during differentiation (14d, continuous exposure) or acutely for 24h after differentiation (acute exposure). Gene expression of lipogenic enzymes and regulating factors will be determined by real‐time PCR. Lipoprotein lipase (LPL) activity was measured using the EnzChek Lipase Substrate and lipolytic rate was determined by measuring glycerol release over 3 hours.ResultsIn differentiated human preadipocytes, a dose‐dependent reduction in LPL activity was observed in both acute and continuous hypoxia (6‐fold reduction under 3% O2 p&lt;0.001, 2‐fold reduction under 10% O2, p&lt;0.05, compared to 21% O2). Basal lipolysis was significantly higher during acute hypoxia (3%) session (p&lt;0.05). No differences in isoproterenol‐ and epinephrine‐stimulated lipolytic responses were observed in subcutaneous abdominal preadipocytes between normoxia and acute hypoxia (3%).ConclusionsThese preliminary results suggest that acute and continuous hypoxia strongly inhibits LPL activity and increases basal lipolysis. These observation supports the hypothesis that hypoxia may adversely affect adipose tissue storage functions, which could in turn contribute to ectopic fat storage, lipotoxicity and a greater prevalence of metabolic disorders in individual with excess adiposity.Support or Funding InformationThis study was supported by the Natural Sciences and Engineering Research Council of Canada as well as the University of Ottawa.