Liver proteomic response to hypertriglyceridemia in human-apolipoprotein C-III transgenic mice at cellular and mitochondrial compartment levels.
ABSTRACT: BACKGROUND: Hypertriglyceridemia (HTG) is defined as a triglyceride (TG) plasma level exceeding 150 mg/dl and is tightly associated with atherosclerosis, metabolic syndrome, obesity, diabetes and acute pancreatitis. The present study was undertaken to investigate the mitochondrial, sub-mitochondrial and cellular proteomic impact of hypertriglyceridemia in the hepatocytes of hypertriglyceridemic transgenic mice (overexpressing the human apolipoproteinC-III). METHODS: Quantitative proteomics (2D-DIGE) analysis was carried out on both "low-expressor" (LE) and "high-expressor" (HE) mice, respectively exhibiting moderate and severe HTG, to characterize the effect of the TG plasma level on the proteomic response. RESULTS: The mitoproteome analysis has revealed a large-scale phenomenon in transgenic mice, i.e. a general down-regulation of matricial proteins and up-regulation of inner membrane proteins. These data also demonstrate that the magnitude of proteomic changes strongly depends on the TG plasma level. Our different analyses indicate that, in HE mice, the capacity of several metabolic pathways is altered to promote the availability of acetyl-CoA, glycerol-3-phosphate, ATP and NADPH for TG de novo biosynthesis. The up-regulation of several cytosolic ROS detoxifying enzymes has also been observed, suggesting that the cytoplasm of HTG mice is subjected to oxidative stress. Moreover, our results suggest that iron over-accumulation takes place in the cytosol of HE mice hepatocytes and may contribute to enhance oxidative stress and to promote cellular proliferation. CONCLUSIONS: These results indicate that the metabolic response to HTG in human apolipoprotein C-III overexpressing mice may support a high TG production rate and that the cytosol of hepatocytes is subjected to an important oxidative stress, probably as a result of FFA over-accumulation, iron overload and enhanced activity of some ROS-producing catabolic enzymes.
Project description:Apolipoprotein A-V (apoA-V), a minor protein associated with lipoproteins, has a major effect on triacylglycerol (TG) metabolism. We investigated whether apoA-V complexed with phospholipid in the form of a reconstituted high-density lipoprotein (rHDL) has potential utility as a therapeutic agent for treatment of hypertriglyceridemia (HTG) when delivered intravenously.Intravenous injection studies were performed in genetically engineered mouse models of severe HTG, including apoav-/- and gpihbp1-/- mice. Administration of apoA-V rHDL to hypertriglyceridemic apoav-/- mice resulted in a 60% reduction in plasma TG concentration after 4 hours. This decline can be attributed to enhanced catabolism/clearance of very-low-density lipoprotein (VLDL), where VLDL TG and cholesterol were reduced ?60%. ApoA-V that associated with VLDL after injection was also rapidly cleared. Site-specific mutations in the heparin-binding region of apoA-V (amino acids 186 to 227) attenuated apoA-V rHDL TG-lowering activity by 50%, suggesting that this sequence element is required for optimal TG-lowering activity in vivo. Unlike apoav-/- mice, injection of apoA-V rHDL into gpihbp1-/- mice had no effect on plasma TG levels, and apoA-V remained associated with plasma VLDL.Intravenously injected apoA-V rHDL significantly lowers plasma TG in an apoA-V deficient mouse model. Its intravenous administration may have therapeutic benefit in human subjects with severe HTG, especially in cases involving apoA-V variants associated with HTG.
Project description:Background:Oxidative stress modulates insulin resistant-related atherogenic dyslipidemia: hypertriglyceridemia (HTG) and low high-density lipoprotein cholesterol (HDL-C) level. Gene polymorphisms in superoxide dismutase (SOD2 and SOD3), glutathione peroxidase-3 (GPX3), and glutathione S-transferase theta-1 (GSTT1) may enable oxidative stress-related lipid abnormalities and severity of coronary atherosclerosis. The present study investigated the associations of antioxidant-related gene polymorphisms with atherogenic dyslipidemia and atherosclerotic severity in subjects with high risk of coronary artery disease (CAD). Methods:Study population comprises of 396 subjects with high risk of CAD. Gene polymorphisms: SOD2 rs4880, SOD3 rs2536512 and rs2855262, GPX rs3828599, and GSTT1 (deletion) were evaluated the associations with HTG, low HDL-C, high TG/HDL-C ratio, and severity of coronary atherosclerosis. Results:SOD2 rs4880-CC, SOD3 rs2536512-AA, rs2855262-CC, and GPX3 rs3828599-AA, but not GSTT1 -/- individually increased risk of HTG combined with low HDL-C level. With a combination of five risk-genotypes as a genetic risk score (GRS), GRS ? 6 increased risks of low HDL-C, high TG/HDL-C ratio, and HTG combined with low HDL-C, comparing with GRS 0-2 [respective adjusted ORs (95% CI) = 2.70 (1.24-5.85), 3.11 (1.55-6.23), and 5.73 (2.22-14.77)]. Gene polymorphisms, though, were not directly associated with severity of coronary atherosclerosis; high TG/HDL-C ratio was associated with coronary atherosclerotic severity [OR = 2.26 (95% CI [1.17-4.34])]. Conclusion:Combined polymorphisms in antioxidant-related genes increased the risk of dyslipidemia related to atherosclerotic severity, suggesting the combined antioxidant-related gene polymorphisms as predictor of atherogenic dyslipidemia.
Project description:BACKGROUND:It is widely agreed that triglyceride (TG)-lowering therapy is imperative in early hypertriglyceridemia-induced acute pancreatitis (HTG-AP). Intravenous insulin with or without heparin, and plasmapheresis are available regimens. However, there is no consensus on first-line therapy. METHODS/DESIGN:The Bi-TPAI trial is a multicenter, parallel group, randomized, controlled, non-inferiority trial in patients with early HTG-AP. The Bi-TPAI trial will include 220 patients with HTG-AP from 17 large tertiary hospitals in China. Patients assigned to the intensive insulin group will be administered an intravenous continuous infusion of regular human insulin at a rate of 0.1?units/kg·h and up to 0.3?units/kg·h. Patients allocated to the plasmapheresis group will receive standard-volume plasmapheresis. The primary endpoint is the time it takes for the TG level to reduce to 500?mg/dl. The secondary endpoints are ICU and hospital lengths of stay, 28-day mortality, severity of HTG-AP, incidence of hypoglycemia, HTG-AP complications, and cost-effectiveness. DISCUSSION:The Bi-TPAI trial will prove that intensive insulin therapy is non-inferior to plasmapheresis. Intensive insulin therapy should be an effective, safe, available, and cheaper triglyceride-lowering therapy for hypertriglyceridemia-induced acute pancreatitis. TRIAL REGISTRATION:ClinicalTrials.gov, NCT03342807 . Registered on 5 Nov 2017.
Project description:We found earlier that apoA-I variants that induced hypertriglyceridemia (HTG) in mice had increased affinity to TG-rich lipoproteins and thereby impaired their catabolism. Here, we tested whether a naturally occurring human apoA-I mutation, Lys107del, associated with HTG also promotes apoA-I binding to TG-rich particles. We expressed apoA-I[Lys107del] variant in Escherichia coli, studied its binding to TG-rich emulsion particles, and performed a physicochemical characterization of the protein. Compared with WT apoA-I, apoA-I[Lys107del] showed enhanced binding to TG-rich particles, lower stability, and greater exposure of hydrophobic surfaces. The crystal structure of truncated, Δ(185-243), apoA-I suggests that deletion of Lys107 disrupts helix registration and disturbs a stabilizing salt bridge network in the N-terminal helical bundle. To elucidate the structural changes responsible for the altered function of apoA-I[Lys107del], we studied another mutant, apoA-I [Lys107Ala]. Our findings suggest that the registry shift and ensuing disruption of the inter-helical salt bridges in apoA-I[Lys107del] result in destabilization of the helical bundle structure and greater exposure of hydrophobic surfaces. We conclude that the structural changes in the apoA-I[Lys107del] variant facilitate its binding to TG-rich lipoproteins and thus, may reduce their lipolysis and contribute to the development of HTG in carriers of the mutation.
Project description:Low-grade inflammation in different tissues, including activation of the nuclear factor ?B pathway in liver, is involved in metabolic disorders such as type 2 diabetes and cardiovascular diseases (CVDs). In this study, we investigated the relation between chronic hepatocyte-specific overexpression of IkB kinase (IKK)-? and hypertriglyceridemia, an important risk factor for CVD, by evaluating whether activation of IKK-? only in the hepatocyte affects VLDL-triglyceride (TG) metabolism directly. Transgenic overexpression of constitutively active human IKK-? specifically in hepatocytes of hyperlipidemic APOE*3-Leiden mice clearly induced hypertriglyceridemia. Mechanistic in vivo studies revealed that the hypertriglyceridemia was caused by increased hepatic VLDL-TG production rather than a change in plasma VLDL-TG clearance. Studies in primary hepatocytes showed that IKK-? overexpression also enhances TG secretion in vitro, indicating a direct relation between IKK-? activation and TG production within the hepatocyte. Hepatic lipid analysis and hepatic gene expression analysis of pathways involved in lipid metabolism suggested that hepatocyte-specific IKK-? overexpression increases VLDL production not by increased steatosis or decreased FA oxidation, but most likely by carbohydrate-responsive element binding protein-mediated upregulation of Fas expression. These findings implicate that specific activation of inflammatory pathways exclusively within hepatocytes induces hypertriglyceridemia. Furthermore, we identify the hepatocytic IKK-? pathway as a possible target to treat hypertriglyceridemia.
Project description:OBJECTIVE:Earlier studies have suggested that a common genetic architecture underlies the clinically heterogeneous polygenic Fredrickson hyperlipoproteinemia (HLP) phenotypes defined by hypertriglyceridemia (HTG). Here, we comprehensively analyzed 504 HLP-HTG patients and 1213 normotriglyceridemic controls and confirmed that a spectrum of common and rare lipid-associated variants underlies this heterogeneity. METHODS AND RESULTS:First, we demonstrated that genetic determinants of plasma lipids and lipoproteins, including common variants associated with plasma triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) from the Global Lipids Genetics Consortium were associated with multiple HLP-HTG phenotypes. Second, we demonstrated that weighted risk scores composed of common TG-associated variants were distinctly increased across all HLP-HTG phenotypes compared with controls; weighted HDL-C and LDL-C risk scores were also increased, although to a less pronounced degree with some HLP-HTG phenotypes. Interestingly, decomposition of HDL-C and LDL-C risk scores revealed that pleiotropic variants (those jointly associated with TG) accounted for the greatest difference in HDL-C and LDL-C risk scores. The APOE E2/E2 genotype was significantly overrepresented in HLP type 3 versus other phenotypes. Finally, rare variants in 4 genes accumulated equally across HLP-HTG phenotypes. CONCLUSIONS:HTG susceptibility and phenotypic heterogeneity are both influenced by accumulation of common and rare TG-associated variants.
Project description:Severe hypertriglyceridemia (HTG) is a relatively common form of dyslipidemia with a complex pathophysiology and serious health complications. HTG can develop in the presence of rare genetic factors disrupting genes involved in the triglyceride (TG) metabolic pathway, including large-scale copy-number variants (CNVs). Improvements in next-generation sequencing technologies and bioinformatic analyses have better allowed assessment of CNVs as possible causes of or contributors to severe HTG. We screened targeted sequencing data of 632 patients with severe HTG and identified partial deletions of the LPL gene, encoding the central enzyme involved in the metabolism of TG-rich lipoproteins, in four individuals (0.63%). We confirmed the genomic breakpoints in each patient with Sanger sequencing. Three patients carried an identical heterozygous deletion spanning the 5' untranslated region (UTR) to LPL exon 2, and one patient carried a heterozygous deletion spanning the 5'UTR to LPL exon 1. All four heterozygous CNV carriers were determined to have multifactorial severe HTG. The predicted null nature of our identified LPL deletions may contribute to relatively higher TG levels and a more severe clinical phenotype than other forms of genetic variation associated with the disease, particularly in the polygenic state. The identification of novel CNVs in patients with severe HTG suggests that methods for CNV detection should be included in the diagnostic workup and molecular genetic evaluation of patients with high TG levels.
Project description:Hypertriglyceridemia (HTG) is a heritable risk factor for cardiovascular disease. Investigating the genetics of HTG may identify new drug targets. There are ~35 known single-nucleotide variants (SNVs) that explain only ~10% of variation in triglyceride (TG) level. Because of the genetic heterogeneity of HTG, a family study design is optimal for identification of rare genetic variants with large effect size because the same mutation can be observed in many relatives and cosegregation with TG can be tested. We considered HTG in a five-generation family of European American descent (n = 121), ascertained for familial combined hyperlipidemia. By using Bayesian Markov chain Monte Carlo joint oligogenic linkage and association analysis, we detected linkage to chromosomes 7 and 17. Whole-exome sequence data revealed shared, highly conserved, private missense SNVs in both SLC25A40 on chr7 and PLD2 on chr17. Jointly, these SNVs explained 49% of the genetic variance in TG; however, only the SLC25A40 SNV was significantly associated with TG (p = 0.0001). This SNV, c.374A>G, causes a highly disruptive p.Tyr125Cys substitution just outside the second helical transmembrane region of the SLC25A40 inner mitochondrial membrane transport protein. Whole-gene testing in subjects from the Exome Sequencing Project confirmed the association between TG and SLC25A40 rare, highly conserved, coding variants (p = 0.03). These results suggest a previously undescribed pathway for HTG and illustrate the power of large pedigrees in the search for rare, causal variants.
Project description:Abstract Background: Hypertriglyceridemia (HTG) is common; however, pseudo-HTG due to high glycerol in glycerol kinase deficiency (GKD, MIM: 307030) is a rare cause of HTG that need to be delineated for appropriate management. GKD is an X-linked recessive disorder characterized by hyperglycerolemia and glyceroluria. Two of three GKD subtypes are known as “isolate” GKD due to a mutation in GK gene alone: (1) symptomatic juvenile form, and (2) benign adult form, associated with an incidental finding of HTG. Since most commercial laboratories determine triglyceride (TG) levels by a glycerol measurement, TG-backbone, patients with GKD are mistakenly labelled as having HTG. Glycerol-blanking is required to reveal the actual TG, but it is costly. Since usual TG-lowering medications are ineffective or even harmful, novel methods to screen for individuals with GKD or pseudo-HTG are necessary. Objective: Through identification of a clinical case of GKD that was diagnosed by glycerol-blanking, we are proposing two potential methods to screen for pseudo-HTG, and presenting their reliability. Methods: The patient was recruited into an IRB-approved study investigating etiologies of dyslipidemia at the University of Pennsylvania. Patient provided consent for medical record review. Results: A 49-year-old man was referred for HTG management. His reported TG levels ranged between 490 and 559 mg/dL without any other adverse lipid levels for several years without a history of pancreatitis or diabetes mellitus. Intriguingly, he reported a family history of HTG. Since TG-lowering medications (fibrates and fish oil) had not reduced his TG levels, specialized lipid analyses were obtained: a non-blanked TG level of 521 mg/dL and a glycerol-blanked TG of 66 mg/dL, consistent with pseudo-HTG or hyperglycerolemia. Repeat glycerol blanked TG levels were 68 and 69 mg/dL, confirming the previous result, and the likely diagnosis of GKD. With two methods, estimated TG levels were calculated, using some of his laboratory values: (1) modified Friedewald equation to solve for TG with a direct LDL (dLDL) value, and (2) the application of a newly developed formula derived from a collection of 17,545 patient samples, to calculate the absolute TG-gap, using apolipoprotein A and B, estimating TG levels (% deltaTG), and determining whether a TG mesurement might be falsely deviated from the “plausible” TG value. Although neither methods showed perfect concordance, the calculated TG-valued derived by the two methods were significantly lower than the non-glycerol blanked TG values. The difference was statistically significant (p<0.05). Conclusion: The patient was clinically diagnosed with GKD, and was taken off of fibrate and the recently added niacin. These two methods can be used quickly to screen for pseudo-HTG or patients with GKD. Currently, it is unknown whether high glycerol levels are associated with high cardiovascular risks.
Project description:Background:Among many causes of hypertriglyceridemia (HTG), familial chylomicronemia syndrome (FCS) is a rare monogenic disorder that manifests as severe HTG and acute pancreatitis. Among the known causal genes for FCS, mutations in APOC2 only account for <2% of cases. Medical nutrition therapy is critical for FCS because usual triglyceride- (TG-) lowering medications are ineffective. Therapeutic plasma exchange (TPE) with fresh frozen plasma (FFP) is an option to urgently reduce TG and pancreatitis episodes. Several novel biologics are under development to treat HTG and may provide therapeutic options for FCS in the future. Objective:We present the challenging care of a 43-year-old man with FCS with apoC-II deficiency and the results of two types of TPE and of investigational TG-lowering biologic therapies. Results:The patient's lipid profile was consistent with FCS. A novel homozygous variant was identified in APOC2, and its pathogenicity was confirmed. Even on a fat-restricted diet, his care was tremendously complicated with unremitting bouts of pancreatitis. TPE with FFP replacement lowered TG >90% post-sessions and appeared to reduce pancreatitis episodes. Experimental ANGPTL3 and APOC3 inhibitors each lowered TG by >50%. Conclusions:Our case demonstrates the importance of delineating and defining the underlying etiology of a rare disorder to optimize therapy and to minimize unfavorable outcomes.