👤 Nathalie Pamir

A 3-year-old patient presented with severe hypertriglyceridemia and suspected familial chylomicronemia syndrome. Genetic analysis of the patient's DNA revealed the presence of 2 different heterozygous nonsense variants in the APOA5 gene encoding apolipoprotein (apo) A-V, namely p.Q275X and p.L242C fs X54. Our objective was to characterize the structural and functional consequences of the patient's co-occuring compound heterozygous variants in APOA5. Biozentrum's SWISS-MODEL was employed to predict the structure of apo A-V variants. Plasma from the patient and their family was used to determine lipid profiles, quantify apo C-II and apo C-III protein levels, and measure lipoprotein lipase (LPL) activity. High-density lipoprotein (HDL) was isolated from plasma and was used to assess sterol efflux capacity and proteome. Structural characterization of the patient's APOA5 variants indicated premature truncation of the C-terminus of apo A-V that comprises the lipid binding domain. The patient's apo A-V was completely absent from the very-low density lipoprotein (VLDL) plasma fraction, associating almost exclusively with the low-density lipoprotein (LDL) and lipoprotein-free fractions. The patient's plasma also demonstrated reduced LPL activity and elevated apo C-II and C-III compared to other family members. The patient's HDL had the lowest sterol efflux capacity of all family members and a distinct proteome with reduced phospholipid transfer protein. Dietary intervention alone was effective in preventing recurring hypertriglyceridemia. These findings add to the current knowledge of apo A-V's role in plasma lipid homeostasis, pointing to a critical role for apo A-V binding to the lipoprotein particle in normal hydrolysis of triglyceride-rich lipoproteins. Show less

ApoB is an essential structural protein for the assembly and secretion of triglyceride-rich lipoproteins and therefore remains a potential target to lower plasma cholesterol levels in hypercholesterolemia patients. To understand the global consequences of APOB gene deficiency, we employed CRISPR-Cas9 system to generate apoB-deficient human hepatoma Huh-7 cells (Ako cells). ApoB was not detectable in the cells and media of the Ako cells. ApoB deficiency had no effect on microsomal triglyceride transfer protein expression and activity. These cells supported apoB48 secretion when transfected with plasmids for the expression of apoB48 suggesting that these cells retain all the lipoprotein assembly and secretion machinery except for apoB expression. APOB gene deficiency had no significant effect on cellular lipid levels, cell growth, and ER stress markers. Proteome analysis of secreted proteins revealed that the most upregulated protein was the vitamin D binding protein, while the most downregulated protein was apoB in Ako cells compared to control cells. This analysis also identified coagulation as an upregulated pathway. Total RNA transcriptome analysis identified DNA replication and complement and coagulation pathways as the most upregulated pathways in Ako cells. Further detailed studies are needed to establish how apoB regulates these pathways. These Ako cells may be useful in studying structure-function analysis of apoB peptides and to address the cellular consequences of disruptions in lipoprotein assembly and secretion. Show less

Prospective cohort studies question the value of HDL-C (high-density lipoprotein cholesterol) for stroke risk prediction. Investigate the relationship between long-term functional recovery and HDL proteome and function. Changes in HDL protein composition and function (cholesterol efflux capacity) in patients after acute ischemic stroke at 2 time points (24 hours, 35 patients; 96 hours, 20 patients) and in 35 control subjects were measured. The recovery from stroke was assessed by 3 months, the National Institutes of Health Stroke Scale and modified Rankin scale scores. When compared with control subject after adjustments for sex and HDL-C levels, 12 proteins some of which participate in acute phase response and platelet activation (APMAP [adipocyte plasma membrane-associated protein], GPLD1 [phosphate inositol-glycan specific phospholipase D], APOE [apolipoprotein E], IHH [Indian hedgehog protein], ITIH4 [inter-alpha-trypsin inhibitor chain H4], SAA2 [serum amyloid A2], APOA4 [apolipoprotein A-IV], CLU [clusterin], ANTRX2 [anthrax toxin receptor 2], PON1 [serum paraoxonase/arylesterase], SERPINA1 [alpha-1-antitrypsin], and APOF [apolipoprotein F]) were significantly (adjusted Changes in HDL proteins during early acute phase of stroke associate with recovery. Monitoring HDL proteins may provide clinical biomarkers that inform on stroke recuperation. Show less

HDLs are nanoparticles with more than 80 associated proteins, phospholipids, cholesterol, and cholesteryl esters. The potential inverse relation of HDL to coronary artery disease (CAD) and the effects of HDL on myriad other inflammatory conditions warrant a better understanding of the genetic basis of the HDL proteome. We conducted a comprehensive genetic analysis of the regulation of the proteome of HDL isolated from a panel of 100 diverse inbred strains of mice (the hybrid mouse diversity panel) and examined protein composition and efflux capacity to identify novel factors that affect the HDL proteome. Genetic analysis revealed widely varied HDL protein levels across the strains. Some of this variation was explained by local Show less

Using genetic and biochemical approaches, we investigated proteins that regulate macrophage cholesterol efflux capacity (CEC) and ABCA1-specific CEC (ABCA1 CEC), 2 functional assays that predict cardiovascular disease (CVD). Macrophage CEC and the concentration of HDL particles were markedly reduced in mice deficient in apolipoprotein A-I (APOA1) or apolipoprotein E (APOE) but not apolipoprotein A-IV (APOA4). ABCA1 CEC was markedly reduced in APOA1-deficient mice but was barely affected in mice deficient in APOE or APOA4. High-resolution size-exclusion chromatography of plasma produced 2 major peaks of ABCA1 CEC activity. The early-eluting peak, which coeluted with HDL, was markedly reduced in APOA1- or APOE-deficient mice. The late-eluting peak was modestly reduced in APOA1-deficient mice but little affected in APOE- or APOA4-deficient mice. Ion-exchange chromatography and shotgun proteomics suggested that plasminogen (PLG) accounted for a substantial fraction of the ABCA1 CEC activity in the peak not associated with HDL. Human PLG promoted cholesterol efflux by the ABCA1 pathway, and PLG-dependent efflux was inhibited by lipoprotein(a) [Lp(a)]. Our observations identify APOA1, APOE, and PLG as key determinants of CEC. Because PLG and Lp(a) associate with human CVD risk, interplay among the proteins might affect atherosclerosis by regulating cholesterol efflux from macrophages. Show less

The lack of high-throughput methods to analyze the adipose tissue protein composition limits our understanding of the protein networks responsible for age and diet related metabolic response. We have developed an approach using multiple-dimension liquid chromatography tandem mass spectrometry and extended multiplexing (24 biological samples) with tandem mass tags (TMT) labeling to analyze proteomes of epididymal adipose tissues isolated from mice fed either low or high fat diet for a short or a long-term, and from mice that aged on low Show less

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL's size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL's content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL's content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL's ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL's APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages. Show less

Apolipoprotein A5 (APOA5) is expressed primarily in the liver and modulates plasma triglyceride levels in mice and humans. Mice overexpressing APOA5 exhibit reduced plasma triglyceride levels. Because there is a tight association between plasma triglyceride concentration and traits of the metabolic syndrome, we used transgenic mice overexpressing human APOA5 to test the concept that these mice would be protected from diet-induced obesity and insulin resistance. Male and female transgenic and wild-type mice on the FVB/N genetic background were fed standard rodent chow or a diet rich in fat and sucrose for 18 weeks, during which time clinical phenotypes associated with obesity and glucose homeostasis were measured. We found that APOA5 transgenic (A5tg) mice were resistant to diet-induced changes in plasma triglyceride but not total cholesterol levels. Body weights were similar between the genotypes for females and males, although male A5tg mice showed a modest but significant increase in the relative size of inguinal fat pads. Although male A5tg mice showed a significantly increased ratio of plasma glucose to insulin, profiles of glucose clearance as evaluated after injections of glucose or insulin failed to reveal any differences between genotypes. Overall, our data showed that there was no advantage to responses to diet-induced obesity with chronic reduction of plasma triglyceride levels as mediated by overexpression of APOA5. Show less

The incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are gut hormones that act via the enteroinsular axis to potentiate insulin secretion from the pancreas in a glucose-dependent manner. Both GLP-1 receptor and GIP receptor knockout mice (GLP-1R(-/-) and GIPR(-/-), respectively) have been generated to investigate the physiological importance of this axis. Although reduced GIP action is a component of type 2 diabetes, GIPR-deficient mice exhibit only moderately impaired glucose tolerance. The present study was directed at investigating possible compensatory mechanisms that take place within the enteroinsular axis in the absence of GIP action. Although serum total GLP-1 levels in GIPR knockout mice were unaltered, insulin responses to GLP-1 from pancreas perfusions and static islet incubations were significantly greater (40-60%) in GIPR(-/-) than in wild-type (GIPR(+/+)) mice. Furthermore, GLP-1-induced cAMP production was also elevated twofold in the islets of the knockout animals. Pancreatic insulin content and gene expression were reduced in GIPR(-/-) mice compared with GIPR(+/+) mice. Paradoxically, immunocytochemical studies showed a significant increase in beta-cell area in the GIPR-null mice but with less intense staining for insulin. In conclusion, GIPR(-/-) mice exhibit altered islet structure and topography and increased islet sensitivity to GLP-1 despite a decrease in pancreatic insulin content and gene expression. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is secreted postprandially and acts in concert with glucose to stimulate insulin secretion from the pancreas. Here, we describe a novel pathway for the regulation of GIP receptor (GIPR) expression within clonal beta-cell lines, pancreatic islets, and in vivo. High (25 mM) glucose was able to significantly reduce GIPR mRNA levels in INS(832/13) cells after only 6 h. In contrast, palmitic acid (2 mM) and WY 14643 (100 microM) stimulated approximate doublings of GIPR expression in INS(832/13) cells under low (5.5 mM), but not high (25 mM), glucose conditions, suggesting that fat can regulate GIPR expression via PPARalpha in a glucose-dependent manner. Both MK-886, an antagonist of PPARalpha, and a dominant negative form of PPARalpha transfected into INS(832/13) cells caused a significant reduction in GIPR expression in low, but not high, glucose conditions. Finally, in hyperglycemic clamped rats, there was a 70% reduction in GIPR expression in the islets and a 71% reduction in GIP-stimulated insulin secretion from the perfused pancreas. Thus, evidence is presented that the GIPR is controlled at normoglycemia by the fatty acid load on the islet; however, when exposed to hyperglycemic conditions, the GIPR is down-regulated, which may contribute to the decreased responsiveness to GIP that is observed in type 2 diabetes. Show less