👤 Guillaume Davidson

Cholesteryl ester transfer protein (CETP) inhibition has been associated with decreased risk of new-onset diabetes in past clinical trials exploring their efficacy in cardiovascular disease and can potentially be repurposed to treat metabolic disease. Notably, as an oral drug it can potentially be used to supplement existing oral drugs such as sodium-glucose cotransporter 2 (SGLT2) inhibitors before patients are required to take injectable drugs such as insulin. To identify whether CETP inhibitors could be used as an oral add-on to SGLT2 inhibition to improve glycemic control. 2×2 factorial Mendelian Randomization (MR) is performed on the general population of UK Biobank participants with European ancestry. Previously constructed genetic scores for CETP and SGLT2 function are combined in a 2×2 factorial framework to characterize the associations between joint CETP and SGLT2 inhibition compared to either alone. Glycated hemoglobin and type-2 diabetes incidence. Data on 233,765 UK Biobank participants suggests that individuals with genetic inhibition of both CETP and SGLT2 have significantly lower glycated hemoglobin levels (mmol/mol) than control (Effect size: -0.136; 95% CI: -0.190 to -0.081; p-value: 1.09E-06), SGLT2 inhibition alone (Effect size: -0.082; 95% CI: -0.140 to -0.024; p-value: 0.00558), and CETP inhibition alone (Effect size: -0.08479; 95% CI: -0.136 to -0.033; p-value: 0.00118). Furthermore, joint CETP and SGLT2 inhibition is associated with decreased incidence of diabetes (log-odds ratio) compared to control (Effect size: -0.068; 95% CI: -0.115 to -0.021; p-value: 4.44E-03) and SGLT2 inhibition alone (Effect size: -0.062; 95% CI: -0.112 to -0.012; p-value: 0.0149). Our results suggest that CETP and SGLT2 inhibitor therapy may improve glycemic control over SGLT2 inhibitors alone. Future clinical trials can explore whether CETP inhibitors can be repurposed to treat metabolic disease and provide an oral therapeutic option to benefit high-risk patients before escalation to injectable drugs such as insulin or glucagon-like peptide 1 (GLP1) receptor agonists. Show less

Obicetrapib, a selective cholesteryl ester transfer protein (CETP) inhibitor, reduces low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol (non-HDL-C), lipoprotein particles, and apolipoproteins, when added to high-intensity statin in patients with dyslipidemia. To evaluate the safety and lipid-altering efficacy of obicetrapib plus ezetimibe combination therapy as an adjunct to high-intensity statin therapy. This double-blind, randomized, phase 2 trial administered 10 mg obicetrapib plus 10 mg ezetimibe (n = 40), 10 mg obicetrapib (n = 39), or placebo (n = 40) for 12 weeks to patients with LDL-C >70 mg/dL and triglycerides (TG) <400 mg/dL, on stable high-intensity statin. Endpoints included concentrations of lipids, apolipoproteins, lipoprotein particles, and proprotein convertase subtilisin kexin type 9 (PCSK9), safety, and tolerability. Ninety-seven patients were included in the primary analysis (mean age 62.6 years, 63.9% male, 84.5% white, average body mass index of 30.9 kg/m The combination of obicetrapib plus ezetimibe significantly lowered atherogenic lipid and lipoprotein parameters, and was safe and well tolerated when administered on top of high-intensity statin to patients with elevated LDL-C. Show less

Low-density lipoprotein (LDL-C) lowering is imperative in cardiovascular disease prevention. We aimed to compare accuracy of three clinically-implemented LDL-C equations in a clinical trial of cholesterol ester transfer protein (CETP) inhibition. Men and women aged 18-75 years with dyslipidaemia were recruited from 17 sites in the Netherlands and Denmark. Patients were randomly assigned to one of nine groups using various combinations of the CETP inhibitor TA-8995 (obicetrapib), statin therapy, and placebo. In pooled measurements over 12 weeks, we calculated LDL-C by the Friedewald, Martin/Hopkins, and Sampson equations, and compared values with preparative ultracentrifugation (PUC) LDL-C overall and with a special interest in the low LDL-C/high triglycerides subgroup. There were 242 patients contributing 921 observations. Overall median LDL-C differences between estimates and PUC were small: Friedewald, 0.00 (25th, 75th: -0.10, 0.08) mmol/L [0 (-4, 3) mg/dL]; Martin/Hopkins, 0.02 (-0.08, 0.10) mmol/L [1 (-3, 4) mg/dL]; and Sampson, 0.05 (-0.03, 0.15) mmol/L [2 (-1, 6) mg/dL]. In the subgroup with estimated LDL-C <1.8 mmol/L (<70 mg/dL) and triglycerides 1.7-4.5 mmol/L (150-399 mg/dL), the Friedewald equation underestimated LDL-C with a median difference versus PUC of -0.25 (-0.33, -0.10) mmol/L [-10 (-13, -4) mg/dL], whereas the median difference by Martin/Hopkins was 0.00 (-0.08, 0.10) mmol/L [0 (-3, 4) mg/dL] and by Sampson was -0.06 (-0.13, 0.00) mmol/L [-2 (-5, 0) mg/dL]. In this subgroup, the proportion of LDL-C observations <1.8 mmol/L (<70 mg/dL) that were correctly classified compared with PUC was 71.4% by Friedewald vs. 100.0% by Martin/Hopkins and 93.1% by Sampson. In European patients with dyslipidaemia receiving a CETP inhibitor, we found improved LDL-C accuracy using contemporary equations vs. the Friedewald equation, and the greatest accuracy was observed with the Martin/Hopkins equation. ClinicalTrials.gov, NCT01970215. Show less

The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS. Show less

Cholesteryl ester transfer protein (CETP) facilitates the exchange of cholesteryl esters and triglycerides (TG) between high-density lipoprotein (HDL) particles and TG-rich, apolipoprotein (apo) B-containing particles. Initially, these compounds were developed to raise plasma HDL cholesterol (HDL-C) levels, a mechanism that was previously thought to lower the risk of atherosclerotic cardiovascular disease (ASCVD). More recently, the focus changed and the use of pharmacologic CETP inhibitors to reduce low-density lipoprotein cholesterol (LDL-C), non-HDL-C and apoB concentrations became supported by several lines of evidence from animal models, observational investigations, randomized controlled trials and Mendelian randomization studies. Furthermore, a cardiovascular outcome trial of anacetrapib demonstrated that CETP inhibition significantly reduced the risk of major coronary events in patients with ASCVD in a manner directly proportional to the substantial reduction in LDL-C and apoB. These data have dramatically shifted the attention on CETP away from raising HDL-C instead to lowering apoB-containing lipoproteins, which is relevant since the newest CETP inhibitor, obicetrapib, reduces LDL-C by up to 51% and apoB by up to 30% when taken in combination with a high-intensity statin. An ongoing cardiovascular outcome trial of obicetrapib in patients with ASCVD is expected to provide further evidence of the ability of CETP inhibitors to reduce major adverse cardiovascular events by lowering apoB. The purpose of the present review is to provide an up-to-date understanding of CETP inhibition and its relationship to ASCVD risk reduction. Show less

Global guidelines for the management of high-cardiovascular-risk patients include aggressive goals for low-density lipoprotein cholesterol (LDL-C). Statin therapy alone is often insufficient to reach goals and nonstatin options have limitations. Here, we tested the lipid-lowering effects of the cholesteryl ester transfer protein (CETP) inhibitor drug obicetrapib in a randomized, double-blind, placebo-controlled trial in dyslipidaemic patients (n = 120, median LDL-C 88 mg dl Show less

Although neurologic symptoms occur in two-thirds of lysosomal storage disorders (LSDs), for most we do not understand the mechanisms underlying brain dysfunction. A major unanswered question is if the pathogenic hallmark of LSDs, storage accumulation, induces functional defects directly or is a disease bystander. Also, for most LSDs we do not know the impact of loss of function in individual cell types. Understanding these critical questions are essential to therapy development. Here, we determine the impact of genetic rescue in distinct cell types on neural circuit dysfunction in CLN3 disease, the most common pediatric dementia and a paradigmatic neurodegenerative LSD. We restored Cln3 expression via AAV-mediated gene delivery and conditional genetic rescue in a CLN3 disease mouse model. Surprisingly, we found that low-level rescue of Cln3 expression in neurons alone normalized clinically relevant electrophysiologic markers of network dysfunction, despite the presence of substantial residual histopathology, in contrast to restoring expression in astrocytes. Thus, loss of CLN3 function in neurons, not storage accumulation, underlies neurologic dysfunction in CLN3 disease. This impliesies that storage clearance may be an inappropriate target for therapy development and an ineffectual biomarker. Show less

The cytoplasmic phosphatase DUSP6 and its nuclear counterpart DUSP5 are negative regulators of RAS/ERK signalling. Here we use deletion of either Dusp5 or Dusp6 to explore the roles of these phosphatases in a murine model of KRAS Show less

BACKGROUND Acute decompensated heart failure (ADHF) is associated with deterioration in renal function-an important risk factor for poor outcomes. Whether ADHF results in permanent kidney damage/dysfunction is unknown. METHODS AND RESULTS We investigated for the first time the renal responses to the development of, and recovery from, ADHF using an ovine model. ADHF development induced pronounced hemodynamic changes, neurohormonal activation, and decline in renal function, including decreased urine, sodium and urea excretion, and creatinine clearance. Following ADHF recovery (25 days), creatinine clearance reductions persisted. Kidney biopsies taken during ADHF and following recovery showed widespread mesangial cell prominence, early mild acute tubular injury, and medullary/interstitial fibrosis. Renal transcriptomes identified altered expression of 270 genes following ADHF development and 631 genes following recovery. A total of 47 genes remained altered post-recovery. Pathway analysis suggested gene expression changes, driven by a network of inflammatory cytokines centered on IL-1β (interleukin 1β), lead to repression of reno-protective eNOS (endothelial nitric oxide synthase) signaling during ADHF development, and following recovery, activation of glomerulosclerosis and reno-protective pathways and repression of proinflammatory/fibrotic pathways. A total of 31 dysregulated genes encoding proteins detectable in urine, serum, and plasma identified potential candidate markers for kidney repair (including Show less

Apolipoprotein A4 (APOA4) is one of the most abundant and versatile apolipoproteins facilitating lipid transport and metabolism. APOA4 is synthesized in the small intestine, packaged onto chylomicrons, secreted into intestinal lymph and transported via circulation to several tissues, including adipose. Since its discovery nearly 4 decades ago, to date, only platelet integrin αIIbβ3 has been identified as APOA4 receptor in the plasma. Using co-immunoprecipitation coupled with mass spectrometry, we probed the APOA4 interactome in mouse gonadal fat tissue, where ApoA4 gene is not transcribed but APOA4 protein is abundant. We demonstrate that lipoprotein receptor-related protein 1 (LRP1) is the cognate receptor for APOA4 in adipose tissue. LRP1 colocalized with APOA4 in adipocytes; it interacted with APOA4 under fasting condition and their interaction was enhanced during lipid feeding concomitant with increased APOA4 levels in plasma. In 3T3-L1 mature adipocytes, APOA4 promoted glucose uptake both in absence and presence of insulin in a dose-dependent manner. Knockdown of LRP1 abrogated APOA4-induced glucose uptake as well as activation of phosphatidylinositol 3 kinase (PI3K)-mediated protein kinase B (AKT). Taken together, we identified LRP1 as a novel receptor for APOA4 in promoting glucose uptake. Considering both APOA4 and LRP1 are multifunctional players in lipid and glucose metabolism, our finding opens up a door to better understand the molecular mechanisms along APOA4-LRP1 axis, whose dysregulation leads to obesity, cardiovascular disease, and diabetes. Show less

OBJECTIVE: Niacin therapy fails to reduce cardiovascular events in statin-treated subjects even though it increases plasma HDL-C (HDL [high-density lipoprotein] cholesterol) and decreases LDL-C (LDL [low-density lipoprotein] cholesterol) and triglyceride levels. To investigate potential mechanisms for this lack of cardioprotection, we quantified the HDL proteome of subjects in 2 niacin clinical trials: the CPC study (Carotid Plaque Composition) and the HDL Proteomics substudy of the AIM-HIGH trial (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides). APPROACH AND RESULTS: Using targeted proteomics, we quantified levels of 31 HDL proteins from 124 CPC subjects and 120 AIM-HIGH subjects. The samples were obtained at baseline and after 1 year of statin monotherapy or niacin-statin combination therapy. Compared with statin monotherapy, niacin-statin combination therapy did not reduce HDL-associated apolipoproteins APOC1, APOC2, APOC3, and APOC4, despite significantly lowering triglycerides. In contrast, niacin markedly elevated HDL-associated PLTP (phospholipid transfer protein), CLU (clusterin), and HP/HPR (haptoglobin/haptoglobinrelated proteins; P≤0.0001 for each) in both the CPC and AIM-HIGH cohorts. CONCLUSIONS: The addition of niacin to statin therapy resulted in elevated levels of multiple HDL proteins linked to increased atherosclerotic risk, which might have compromised the cardioprotective effects associated with higher HDL-C levels and lower levels of LDL-C and triglycerides. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00715273; NCT00880178; NCT00120289. Show less

Optimal maternal long-chain PUFA (LCPUFA) status is essential for the developing fetus. The fatty acid desaturase (FADS) genes are involved in the endogenous synthesis of LCPUFA. The minor allele of various FADS SNP have been associated with increased maternal concentrations of the precursors linoleic acid (LA) and α-linolenic acid (ALA), and lower concentrations of arachidonic acid (AA) and DHA. There is limited research on the influence of FADS genotype on cord PUFA status. The current study investigated the influence of maternal and child genetic variation in FADS genotype on cord blood PUFA status in a high fish-eating cohort. Cord blood samples (n 1088) collected from the Seychelles Child Development Study (SCDS) Nutrition Cohort 2 (NC2) were analysed for total serum PUFA. Of those with cord PUFA data available, maternal (n 1062) and child (n 916), FADS1 (rs174537 and rs174561), FADS2 (rs174575), and FADS1-FADS2 (rs3834458) were determined. Regression analysis determined that maternal minor allele homozygosity was associated with lower cord blood concentrations of DHA and the sum of EPA + DHA. Lower cord blood AA concentrations were observed in children who were minor allele homozygous for rs3834458 (β = 0·075; P = 0·037). Children who were minor allele carriers for rs174537, rs174561, rs174575 and rs3834458 had a lower cord blood AA:LA ratio (P < 0·05 for all). Both maternal and child FADS genotype were associated with cord LCPUFA concentrations, and therefore, the influence of FADS genotype was observed despite the high intake of preformed dietary LCPUFA from fish in this population. Show less

The aim of this study was to analyze the biological role of different transforming growth factor-β (TGFβ) receptor splice variants in ovarian carcinoma (OC). Specific receptor variant knockouts (KO) were prepared using the CRISPR/Cas9 genome editing system in two OC cell lines, TβRI variant 1 (TβRIv1) KO in ES-2 cells and TβRII variant 1 (TβRIIv1) KO in OVCAR-8 cells. Control and KO cells were compared by proteomic analysis, functional tests, analysis of epithelial-mesenchymal transition (EMT) drivers, and Western blot of signaling proteins. Proteomic analysis revealed significant changes in protein pathways in the KO cells. TβRIv1 KO resulted in a significant reduction in both cellular motility and invasion, while TβRIIv1 KO significantly reduced cellular motility and increased Reactive Oxygen Species (ROS) production. Both receptor variant KOs reduced MET protein levels. Of the EMT drivers, a significant decrease in TWIST protein expression, and increase in SNAIL protein and Show less

The human genome encodes thousands of non-coding RNAs. Many of these terminate early and are then rapidly degraded, but how their transcription is restricted is poorly understood. In a screen for protein-coding gene transcriptional termination factors, we identified ZC3H4. Its depletion causes upregulation and extension of hundreds of unstable transcripts, particularly antisense RNAs and those transcribed from so-called super-enhancers. These loci are occupied by ZC3H4, suggesting that it directly functions in their transcription. Consistently, engineered tethering of ZC3H4 to reporter RNA promotes its degradation by the exosome. ZC3H4 is predominantly metazoan -interesting when considering its impact on enhancer RNAs that are less prominent in single-celled organisms. Finally, ZC3H4 loss causes a substantial reduction in cell proliferation, highlighting its overall importance. In summary, we identify ZC3H4 as playing an important role in restricting non-coding transcription in multicellular organisms. Show less

Apolipoprotein A-IV (ApoA-IV) synthesized by the gut regulates lipid metabolism. Sympathetic innervation of adipose tissues also controls lipid metabolism. We hypothesized that ApoA-IV required sympathetic innervation to increase fatty acid (FA) uptake by adipose tissues and brown adipose tissue (BAT) thermogenesis. After 3 weeks feeding of either a standard chow diet or a high-fat diet (HFD), mice with unilateral denervation of adipose tissues received intraperitoneal administration of recombinant ApoA-IV protein and intravenous infusion of lipid mixture with radioactive triolein. In chow-fed mice, ApoA-IV administration increased FA uptake by intact BAT but not the contralateral denervated BAT or intact white adipose tissue (WAT). Immunoblots showed that, in chow-fed mice, ApoA-IV increased expression of lipoprotein lipase and tyrosine hydroxylase in both intact BAT and inguinal WAT (IWAT), while ApoA-IV enhanced protein levels of β3 adrenergic receptor, adipose triglyceride lipase, and uncoupling protein 1 in the intact BAT only. In HFD-fed mice, ApoA-IV elevated FA uptake by intact epididymal WAT (EWAT) but not intact BAT or IWAT. ApoA-IV increased sympathetic activity assessed by norepinephrine turnover (NETO) rate in BAT and EWAT of chow-fed mice, whereas it elevated NETO only in EWAT of HFD-fed mice. These observations suggest that, in chow-fed mice, ApoA-IV activates sympathetic activity of BAT and increases FA uptake by BAT via innervation, while in HFD-fed mice, ApoA-IV stimulates sympathetic activity of EWAT to shunt FAs into the EWAT. Show less

APOA5 is a low-abundance exchangeable apolipoprotein that plays critical roles in human triglyceride (TG) metabolism. Indeed, aberrations in the plasma concentration or structure of APOA5 are linked to hypertriglyceridemia, hyperchylomicronemia, myocardial infarction risk, obesity, and coronary artery disease. While it has been successfully produced at low yield in bacteria, the resulting protein had limitations for structure-function studies due to its low solubility under physiological buffer conditions. We hypothesized that the yield and solubility of recombinant APOA5 could be increased by: Show less

Lipoprotein particles with abnormal compositions, such as lipoprotein X (LP-X) and lipoprotein Z (LP-Z), have been described in cases of obstructive jaundice and cholestasis. The study objectives were to: (1) develop an NMR-based assay for quantification of plasma/serum LP-Z particles, (2) evaluate the assay performance, (3) isolate LP-Z particles and characterize them by lipidomic and proteomic analysis, and (4) quantify LP-Z in subjects with various liver diseases. Assay performance was assessed for linearity, sensitivity, and precision. Mass spectroscopy was used to characterize the protein and lipid content of isolated LP-Z particles. The assay showed good linearity and precision (2.5-6.3%). Lipid analyses revealed that LP-Z particles exhibit lower cholesteryl esters and higher free cholesterol, bile acids, acylcarnitines, diacylglycerides, dihexosylceramides, lysophosphatidylcholines, phosphatidylcholines, triacylglycerides, and fatty acids than low-density lipoprotein (LDL) particles. A proteomic analysis revealed that LP-Z have one copy of apolipoprotein B per particle such as LDL, but less apolipoprotein (apo)A-I, apoC3, apoA-IV and apoC2 and more complement C3. LP-Z were not detected in healthy volunteers or subjects with primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, or type 2 diabetes. LP-Z were detected in some, but not all, subjects with hypertriglyceridemia, and were high in some subjects with alcoholic liver disease. LP-Z differ significantly in their lipid and protein content from LDL. Further studies are needed to fully understand the pathophysiological reason for the enhanced presence of LP-Z particles in patients with cholestasis and alcoholic liver disease. Show less

Maternal status of long-chain PUFAs (LC-PUFAs) may be related to fetal growth. Maternal fish consumption exposes the mother to the neurotoxicant methylmercury (MeHg), which, in contrast, may restrict fetal growth. Our aim was to examine relations between maternal LC-PUFA status at 28 wk and birth outcomes (birth weight, length, and head circumference), controlling for MeHg exposure throughout pregnancy, in the Seychelles Child Development Study Nutrition Cohort 2. Our secondary aim was to examine the influence of maternal variation in genes regulating the desaturation of LC-PUFAs [fatty acid desaturase (FADS)] on birth outcomes. From nonfasting blood samples collected at 28 wk of gestation, we measured serum total LC-PUFA concentrations and FADS1 (rs174537, rs174561), FADS1-FADS2rs3834458, and FADS2rs174575 genotypes, with hair total mercury concentrations assessed at delivery. Data were available for n = 1236 mother-child pairs. Associations of maternal LC-PUFAs, MeHg, and FADS genotype with birth outcomes were assessed by multiple linear regression models, adjusting for child sex, gestational age, maternal age, BMI, alcohol use, socioeconomic status, and parity. In our cohort of healthy mothers, neither maternal LC-PUFA status nor MeHg exposure were significant determinants of birth outcomes. However, when compared with major allele homozygotes, mothers who were heterozygous for the minor allele of FADS1 (rs174537 and rs174561, GT compared with TT, β = 0.205, P = 0.03; TC compared with CC, β = 0.203, P = 0.04) and FADS1-FADS2 (rs3834458, Tdel compared with DelDel, β = 0.197, P = 0.04) had infants with a greater head circumference (all P < 0.05). Homozygosity for the minor allele of FADS2 (rs174575) was associated with a greater birth weight (GG compared with CC, β = 0.109, P = 0.04). In our mother-child cohort, neither maternal LC-PUFA status nor MeHg exposure was associated with birth outcomes. The observed associations of variation in maternal FADS genotype with birth outcomes should be confirmed in other populations. Show less

Accumulation of lysosomal storage material and late-stage neurodegeneration are hallmarks of lysosomal storage disorders (LSDs) affecting the brain. Yet, for most LSDs, including CLN3 disease, the most common form of childhood dementia, it is unclear what mechanisms drive neurologic symptoms. Do deficits arise from loss of function of the mutated protein or toxicity from storage accumulation? Here, using in vitro voltage-sensitive dye imaging and in vivo electrophysiology, we find progressive hippocampal dysfunction occurs before notable lysosomal storage and neuronal loss in 2 CLN3 disease mouse models. Pharmacologic reversal of lysosomal storage deposition in young mice does not rescue this circuit dysfunction. Additionally, we find that CLN3 disease mice lose an electrophysiologic marker of new memory encoding - hippocampal sharp-wave ripples. This discovery, which is also seen in Alzheimer's disease, suggests the possibility of a shared electrophysiologic signature of dementia. Overall, our data describe new insights into previously unknown network-level changes occurring in LSDs affecting the central nervous system and highlight the need for new therapeutic interventions targeting early circuit defects. Show less

Platelet αIIbβ3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet β3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbβ3 integrin, and the direct apoA-IV-αIIbβ3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbβ3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbβ3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases. Show less

Cholecystokinin (CCK) and apolipoprotein A-IV (ApoA-IV) are gastrointestinal peptides that play an important role in controlling energy homeostasis. Lymphatic ApoA-IV and plasma CCK secretion are mediated via a chylomicron formation-dependent pathway during a dietary lipid infusion. Given their similar roles as satiating proteins, the present study examines how the two peptides interact in their function. Specifically, this study sought to understand how ApoA-IV regulates CCK secretion. For this purpose, Cck gene expression in the small intestines of ApoA-IV knockout (ApoA-IV-KO) and wild-type (WT) mice were compared under an array of feeding conditions. When fed with a chow or high-fat diet (HFD), basal levels of Cck transcripts were significantly reduced in the duodenum of ApoA-IV-KO mice compared to WT mice. Furthermore, after an oral gavage of a lipid mixture, Cck gene expression in the duodenum was significantly reduced in ApoA-IV-KO mice relative to the change seen in WT mice. To determine the mechanism by which ApoA-IV modulates Cck gene expression, STC-1 cells were transfected with predesigned mouse lysophosphatidic acid receptor 5 (LPAR5) small interfering RNA (siRNA) to knockdown Lpar5 gene expression. In this in-vitro study, mouse recombinant ApoA-IV protein increased Cck gene expression in enteroendocrine STC-1 cells and stimulated CCK release from the STC-1 cells. However, the levels of CCK protein and Cck expression were attenuated when Lpar5 was knocked down in the STC-1 cells. Together these observations suggest that dietary lipid-induced ApoA-IV is associated with Cck synthesis in the duodenum and that ApoA-IV protein directly enhances CCK release through the activation of a LPAR5-dependent pathway. Show less

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced autofluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use. Show less

Given their association with cardiovascular disease protection, there has been intense interest in understanding the biology of high density lipoproteins (HDL). HDL is actually a family of diverse particle types, each made up of discrete - but as yet undetermined - combinations of proteins drawn from up to 95 lipophilic plasma proteins. The abundant apolipoproteins (apo) of the A class (apoA-I, apoA-II and apoA-IV) have been proposed to act as organizing platforms for auxiliary proteins, but this concept has not been systematically evaluated. We assessed the impact of genetic knock down of each platform protein on the particle size distribution of auxiliary HDL proteins. Loss of apoA-I or apoA-II massively reduced HDL lipids and changed the plasma size pattern and/or abundance of several plasma proteins. Surprisingly though, many HDL proteins were not affected, suggesting they assemble on lipid particles in the absence of apoA-I or apoA-II. In contrast, apoA-IV ablation had minor effects on plasma lipids and proteins, suggesting that it forms particles that largely exclude other apolipoproteins. Overall, the data indicate that distinct HDL subpopulations exist that do not contain, nor depend on, apoA-I, apoA-II or apoA-IV and these contribute substantially to the proteomic diversity of HDL. Plasma levels of high density lipoproteins (HDL) are inversely correlated with cardiovascular disease. These particles are becoming known as highly heterogeneous entities that have diverse compositions and functions that may impact disease. Unfortunately, we know little about the forces that maintain the composition of each particle in plasma. It has been suggested that certain 'scaffold' proteins, such as apolipoprotein (apo) A-I, apoA-II and apoA-IV, may act as organizing centers for the docking of myriad accessory proteins. To test this hypothesis, we took advantage of the genetic tractability of the mouse model and ablated these three proteins individually. We then tracked the abundance and size profile of the remaining HDL proteins by gel filtration chromatography combined with mass spectrometry. The results clearly show that certain cohorts of proteins depend on each scaffold molecule to assemble normal sized HDL particles under wild-type conditions. This work forms the basis for more detailed studies that will define the specific compositions of HDL subspecies with the possibility of connecting them to specific functions or roles in disease. Show less

Myotubular myopathy (MTM) is a devastating pediatric neuromuscular disorder of phosphoinositide (PIP) metabolism resulting from mutations of the PIP phosphatase MTM1 for which there are no treatments. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in animal models of MTM. Here, we tested the hypothesis that lowering PI3P levels may prevent or reverse the MTM disease process. To test this, we targeted class II and III PI3 kinases (PI3Ks) in an MTM1-deficient mouse model. Muscle-specific ablation of Pik3c2b, but not Pik3c3, resulted in complete prevention of the MTM phenotype, and postsymptomatic targeting promoted a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that certain PI3K inhibitors prevented development of the zebrafish mtm phenotype. Finally, the PI3K inhibitor wortmannin improved motor function and prolonged lifespan of the Mtm1-deficient mice. In all, we have identified Pik3c2b as a genetic modifier of Mtm1 mutation and demonstrated that PIK3C2B inhibition is a potential treatment strategy for MTM. In addition, we set the groundwork for similar reciprocal inhibition approaches for treating other PIP metabolic disorders and highlight the importance of modifier gene pathways as therapeutic targets. Show less

We have previously shown that the nuclear receptor, NR1D1, is a cofactor in ApoA-IV-mediated downregulation of gluconeogenesis. Nuclear receptor, NR4A1, is involved in the transcriptional regulation of various genes involved in inflammation, apoptosis, and glucose metabolism. We investigated whether NR4A1 influences the effect of ApoA-IV on hepatic glucose metabolism. Our in situ proximity ligation assays and coimmunoprecipitation experiments indicated that ApoA-IV colocalized with NR4A1 in human liver (HepG2) and kidney (HEK-293) cell lines. The chromatin immunoprecipitation experiments and luciferase reporter assays indicated that the ApoA-IV and NR4A1 colocalized at the RORα response element of the human G6Pase promoter, reducing its transcriptional activity. Our RNA interference experiments showed that knocking down the expression of NR4A1 in primary mouse hepatocytes treated with ApoA-IV increased the expression of NR1D1, G6Pase, and PEPCK, and that knocking down NR1D1 expression increased the level of NR4A1. We also found that ApoA-IV induced the expression of endogenous NR4A1 in both cultured primary mouse hepatocytes and in the mouse liver, and decreased glucose production in primary mouse hepatocytes. Our findings showed that ApoA-IV colocalizes with NR4A1, which suppresses G6Pase and PEPCK gene expression at the transcriptional level, reducing hepatic glucose output and lowering blood glucose. The ApoA-IV-induced increase in NR4A1 expression in hepatocytes mediates further repression of gluconeogenesis. Our findings suggest that NR1D1 and NR4A1 serve similar or complementary functions in the ApoA-IV-mediated regulation of gluconeogenesis. Show less

Apolipoprotein (apo)A-IV is a lipid emulsifying protein linked to a range of protective roles in obesity, diabetes, and cardiovascular disease. It exists in several states in plasma including lipid-bound in HDL and chylomicrons and as monomeric and dimeric lipid-free/poor forms. Our recent x-ray crystal structure of the central domain of apoA-IV shows that it adopts an elongated helical structure that dimerizes via two long reciprocating helices. A striking feature is the alignment of conserved proline residues across the dimer interface. We speculated that this plays important roles in the structure of the lipid-free protein and its ability to bind lipid. Here we show that the systematic conversion of these prolines to alanine increased the thermodynamic stability of apoA-IV and its propensity to oligomerize. Despite the structural stabilization, we noted an increase in the ability to bind and reorganize lipids and to promote cholesterol efflux from cells. The novel properties of these mutants allowed us to isolate the first trimeric form of an exchangeable apolipoprotein and characterize it by small-angle x-ray scattering and chemical cross-linking. The results suggest that the reciprocating helix interaction is a common feature of all apoA-IV oligomers. We propose a model of how self-association of apoA-IV can result in spherical lipoprotein particles, a model that may have broader applications to other exchangeable apolipoprotein family members. Show less

Long-chain n-6 and n-3 PUFA (LC-PUFA), arachidonic acid (AA) (20:4n-6) and DHA (22:6n-3), are critical for optimal brain development. These fatty acids can be consumed directly from the diet, or synthesized endogenously from precursor PUFA by Δ-5 (encoded by FADS1) and Δ-6 desaturases (encoded by FADS2). The aim of this study was to determine the potential importance of maternal genetic variability in FADS1 and FADS2 genes to maternal LC-PUFA status and infant neurodevelopment in populations with high fish intakes. The Nutrition Cohorts 1 (NC1) and 2 (NC2) are longitudinal observational mother-child cohorts in the Republic of Seychelles. Maternal serum LC-PUFA was measured at 28 weeks gestation and genotyping for rs174537 (FADS1), rs174561 (FADS1), rs3834458 (FADS1-FADS2) and rs174575 (FADS2) was performed in both cohorts. The children completed the Bayley Scales of Infant Development II (BSID-II) at 30 months in NC1 and at 20 months in NC2. Complete data were available for 221 and 1310 mothers from NC1 and NC2 respectively. With increasing number of rs3834458 minor alleles, maternal concentrations of AA were significantly decreased (NC1 p=0.004; NC2 p<0.001) and precursor:product ratios for linoleic acid (LA) (18:2n-6)-to-AA (NC1 p<0.001; NC2 p<0.001) and α-linolenic acid (ALA) (18:3n-3)-to-DHA were increased (NC2 p=0.028). There were no significant associations between maternal FADS genotype and BSID-II scores in either cohort. A trend for improved PDI was found among infants born to mothers with the minor rs3834458 allele.In these high fish-eating cohorts, genetic variability in FADS genes was associated with maternal AA status measured in serum and a subtle association of the FADS genotype was found with neurodevelopment. Show less

Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including anaphase-promoting complex/cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC's RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2-ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain-elongating E2 UBE2S in ways that differ from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation. Show less

Apolipoprotein AIV (Apo AIV) and cholecystokinin (CCK) are secreted in response to fat consumption, and both cause satiation via CCK 1 receptor (CCK-1R)-containing vagal afferent nerves to the nucleus of the solitary tract (NTS), where Apo AIV is also synthesized. Fasted male Long-Evans rats received ip CCK-8 or fourth-ventricular (i4vt) Apo AIV alone or in combination. Food intake and c-Fos proteins (a product of the c-Fos immediate-early gene) were assessed. i4vt Apo AIV and/or ip CCK at effective doses reduced food intake and activated c-Fos proteins in the NTS and hypothalamic arcuate nucleus and paraventricular nucleus. Blockade of the CCK-1R by i4vt lorglumide adjacent to the NTS attenuated the satiating and c-Fos-stimulating effects of CCK and Apo AIV, alone or in combination. Maintenance on a high-fat diet (HFD) for 10 weeks resulted in weight gain and attenuation of both the behavioral and c-Fos responses to a greater extent than occurred in low-fat diet-fed and pair-fed HFD animals. These observations suggest that NTS Apo AIV or/and peripheral CCK requires vagal CCK-1R signaling to elicit satiation and that maintenance on a HFD reduces the satiating capacity of these 2 signals. Show less