Lipid Droplet-Derived Monounsaturated Fatty Acids Traffic via PLIN5 to Allosterically Activate SIRT1.

Lipid droplets (LDs) provide a reservoir for triacylglycerol storage and are a central hub for fatty acid trafficking and signaling in cells. Lipolysis promotes mitochondrial biogenesis and oxidative metabolism via a SIRT1/PGC-1α/PPARα-dependent pathway through an unknown mechanism. Herein, we identify that monounsaturated fatty acids (MUFAs) allosterically activate SIRT1 toward select peptide-substrates such as PGC-1α. MUFAs enhance PGC-1α/PPARα signaling and promote oxidative metabolism in cells and animal models in a SIRT1-dependent manner. Moreover, we characterize the LD protein perilipin 5 (PLIN5), which is known to enhance mitochondrial biogenesis and function, to be a fatty-acid-binding protein that preferentially binds LD-derived monounsaturated fatty acids and traffics them to the nucleus following cAMP/PKA-mediated lipolytic stimulation. Thus, these studies identify the first-known endogenous allosteric modulators of SIRT1 and characterize a LD-nuclear signaling axis that underlies the known metabolic benefits of MUFAs and PLIN5.

Global quantitative proteomic analysis of aged mouse hippocampus.

Understanding the molecular changes associated with the aged brain forms the basis for developing potential strategies for slowing cognitive decline associated with normal aging. Focusing on the hippocampus, a critical brain region involved in learning and memory, we employed tandem mass tag methodology to investigate global proteomic changes that occur in advanced-aged (20-month) versus young (3-month) C57BL/6 male mice. Our analysis revealed the upregulation of 236 proteins in the old hippocampal proteome, including those enriched within several age-related processes, such as the adaptive immune response and molecular metabolic pathways, whereas downregulated proteins (88 in total) are mainly involved in axonogenesis and growth cone-related processes. Categorizing proteins by cell-type enrichment in the brain identified a general upregulation of proteins preferentially expressed in microglia, astrocytes, and oligodendrocytes. In contrast, proteins with neuron-specific expression displayed an overall age-related downregulation. By integrating our proteomic with our previously published transcriptomic data, we discovered a mild but significant positive correlation between mRNA and protein expression changes in the aged hippocampus. Therefore, this proteomic data is a valuable additional resource for further understanding age-related molecular mechanisms.

Insulin Prevents Hypercholesterolemia by Suppressing 12α-Hydroxylated Bile Acids.

BACKGROUND: The risk of cardiovascular disease in type 1 diabetes remains extremely high, despite marked advances in blood glucose control and even the widespread use of cholesterol synthesis inhibitors. Thus, a deeper understanding of insulin regulation of cholesterol metabolism, and its disruption in type 1 diabetes, could reveal better treatment strategies. METHODS: To define the mechanisms by which insulin controls plasma cholesterol levels, we knocked down the insulin receptor, FoxO1, and the key bile acid synthesis enzyme, CYP8B1. We measured bile acid composition, cholesterol absorption, and plasma cholesterol. In parallel, we measured markers of cholesterol absorption and synthesis in humans with type 1 diabetes treated with ezetimibe and simvastatin in a double-blind crossover study. RESULTS: Mice with hepatic deletion of the insulin receptor showed marked increases in 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol. This phenotype was entirely reversed by hepatic deletion of FoxO1. FoxO1 is inhibited by insulin and required for the production of 12α-hydroxylated bile acids, which promote intestinal cholesterol absorption and suppress hepatic cholesterol synthesis. Knockdown of Cyp8b1 normalized 12α-hydroxylated bile acid levels and completely prevented hypercholesterolemia in mice with hepatic deletion of the insulin receptor (n=5-30), as well as mouse models of type 1 diabetes (n=5-22). In parallel, the cholesterol absorption inhibitor, ezetimibe, normalized cholesterol absorption and low-density lipoprotein cholesterol in patients with type 1 diabetes as well as, or better than, the cholesterol synthesis inhibitor, simvastatin (n=20). CONCLUSIONS: Insulin, by inhibiting FoxO1 in the liver, reduces 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol levels. Thus, type 1 diabetes leads to a unique set of derangements in cholesterol metabolism, with increased absorption rather than synthesis. These derangements are reversed by ezetimibe, but not statins, which are currently the first line of lipid-lowering treatment in type 1 diabetes. Taken together, these data suggest that a personalized approach to lipid lowering in type 1 diabetes may be more effective and highlight the need for further studies specifically in this group of patients.

A role for NPY-NPY2R signaling in albuminuric kidney disease.

Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that Neuropeptide Y (NPY) was significantly down-regulated in insulin-resistant vs. insulin-sensitive mouse podocytes and in human glomeruli of patients with early and late-stage diabetic nephropathy, as well as other nondiabetic glomerular diseases. This contrasts with the increased plasma and urinary levels of NPY that are observed in such conditions. Studying NPY-knockout mice, we found that NPY deficiency in vivo surprisingly reduced the level of albuminuria and podocyte injury in models of both diabetic and nondiabetic kidney disease. In vitro, podocyte NPY signaling occurred via the NPY2 receptor (NPY2R), stimulating PI3K, MAPK, and NFAT activation. Additional unbiased proteomic analysis revealed that glomerular NPY-NPY2R signaling predicted nephrotoxicity, modulated RNA processing, and inhibited cell migration. Furthermore, pharmacologically inhibiting the NPY2R in vivo significantly reduced albuminuria in adriamycin-treated glomerulosclerotic mice. Our findings suggest a pathogenic role of excessive NPY-NPY2R signaling in the glomerulus and that inhibiting NPY-NPY2R signaling in albuminuric kidney disease has therapeutic potential.

Migration and Migrant Labour in the Gig Economy: An Intervention.

In urban gig economies around the world, platform labour is predominantly migrant labour, yet research on the intersection of the gig economy and labour migration remains scant. Our experience with two action research projects, spanning six cities on four continents, has taught us how platform work impacts the structural vulnerability of migrant workers. This leads us to two claims that should recalibrate the gig economy research agenda. First, we argue that platform labour simultaneously degrades working conditions while offering migrants much-needed opportunities to improve their livelihoods. Second, we contend that the reclassification of gig workers as employees is by itself not sufficient to counter the precarisation of migrant gig work. Instead, we need ambitious policies at the intersection of immigration, social welfare, and employment regulation that push back against the digitally mediated commodification of migrant labour worldwide.

The temporal profile of activity-dependent presynaptic phospho-signalling reveals long-lasting patterns of poststimulus regulation.

Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic activity-dependent phospho-signalling, which includes two KCl stimulation levels and analysis of the poststimulus period. We profiled 1,917 regulated phosphopeptides and bioinformatically identified six temporal patterns of co-regulated proteins. The presynaptic proteins with large changes in phospho-status were again prominently regulated in the analysis of 7,070 activity-dependent phosphopeptides from KCl-stimulated cultured hippocampal neurons. Active zone scaffold proteins showed a high level of activity-dependent phospho-regulation that far exceeded the response from postsynaptic density scaffold proteins. Accordingly, bassoon was identified as the major target of neuronal phospho-signalling. We developed a probabilistic computational method, KinSwing, which matched protein kinase substrate motifs to regulated phosphorylation sites to reveal underlying protein kinase activity. This approach allowed us to link protein kinases to profiles of co-regulated presynaptic protein networks. Ca2+- and calmodulin-dependent protein kinase IIα (CaMKIIα) responded rapidly, scaled with stimulus strength, and had long-lasting activity. Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) was the main protein kinase predicted to control a distinct and significant pattern of poststimulus up-regulation of phosphorylation. This work provides a unique resource of activity-dependent phosphorylation sites of synaptosomes and neurons, the vast majority of which have not been investigated with regard to their functional impact. This resource will enable detailed characterization of the phospho-regulated mechanisms impacting the plasticity of neurotransmitter release.

Pathway Home™ for High Utilizers of Psychiatric Inpatient Services: Impact on Inpatient Days and Outpatient Engagement.

OBJECTIVE: This study examined the impact of Pathway Home™ (PH) transition services for high utilizers of psychiatric hospitalization on inpatient days and outpatient engagement post-hospital discharge. METHODS: This case series study of forty PH graduates (5/22/2015-8/31/2018) used Medicaid claims to assess psychiatric inpatient days-per-month, average proportion of months with psychiatric emergency room, outpatient, and health home care management services. T-tests compared three time periods: the year prior, during, and after enrollment. RESULTS: Graduates had significantly fewer psychiatric inpatient days/month during (M = 1.84, p < 0.001) and after PH enrollment (M = 1.88, p < 0.001) compared to prior to enrollment (M = 7.1), while emergency services were stable. Outpatient visits increased from 45% prior to 76% during enrollment (p < 0.001) and was sustained on follow-up (67%, p = 0.008). A similar pattern emerged for health home services (32%, 60%, and 50%). CONCLUSION: PH is a promising approach for improving outcomes for high utilizers of psychiatric inpatient services, with sustained impact on follow-up.

Diet quality and risk and severity of COVID-19: a prospective cohort study.

OBJECTIVE: Poor metabolic health and unhealthy lifestyle factors have been associated with risk and severity of COVID-19, but data for diet are lacking. We aimed to investigate the association of diet quality with risk and severity of COVID-19 and its interaction with socioeconomic deprivation. DESIGN: We used data from 592 571 participants of the smartphone-based COVID-19 Symptom Study. Diet information was collected for the prepandemic period using a short food frequency questionnaire, and diet quality was assessed using a healthful Plant-Based Diet Score, which emphasises healthy plant foods such as fruits or vegetables. Multivariable Cox models were fitted to calculate HRs and 95% CIs for COVID-19 risk and severity defined using a validated symptom-based algorithm or hospitalisation with oxygen support, respectively. RESULTS: Over 3 886 274 person-months of follow-up, 31 815 COVID-19 cases were documented. Compared with individuals in the lowest quartile of the diet score, high diet quality was associated with lower risk of COVID-19 (HR 0.91; 95% CI 0.88 to 0.94) and severe COVID-19 (HR 0.59; 95% CI 0.47 to 0.74). The joint association of low diet quality and increased deprivation on COVID-19 risk was higher than the sum of the risk associated with each factor alone (Pinteraction=0.005). The corresponding absolute excess rate per 10 000 person/months for lowest vs highest quartile of diet score was 22.5 (95% CI 18.8 to 26.3) among persons living in areas with low deprivation and 40.8 (95% CI 31.7 to 49.8) among persons living in areas with high deprivation. CONCLUSIONS: A diet characterised by healthy plant-based foods was associated with lower risk and severity of COVID-19. This association may be particularly evident among individuals living in areas with higher socioeconomic deprivation.

Reversal of hypertriglyceridemia in diabetic BTBR ob/ob mice does not prevent nephropathy.

The etiology of diabetic nephropathy in type 2 diabetes is multifactorial. Sustained hyperglycemia is a major contributor, but additional contributions come from the hypertension, obesity, and hyperlipidemia that are also commonly present in patients with type 2 diabetes and nephropathy. The leptin deficient BTBR ob/ob mouse is a model of type 2 diabetic nephropathy in which hyperglycemia, obesity, and hyperlipidemia, but not hypertension, are present. We have shown that reversal of the constellation of these metabolic abnormalities with leptin replacement can reverse the morphologic and functional manifestations of diabetic nephropathy. Here we tested the hypothesis that reversal specifically of the hypertriglyceridemia, using an antisense oligonucleotide directed against ApoC-III, an apolipoprotein that regulates the interactions of VLDL (very low density lipoproteins) with the LDL receptor, is sufficient to ameliorate the nephropathy of Type 2 diabetes. Antisense treatment resulted in reduction of circulating ApoC-III protein levels and resulted in substantial lowering of triglycerides to near-normal levels in diabetic mice versus controls. Antisense treatment did not ameliorate proteinuria or pathologic manifestations of diabetic nephropathy, including podocyte loss. These studies indicate that pathologic manifestations of diabetic nephropathy are unlikely to be reduced by lipid-lowering therapeutics alone, but does not preclude a role for such interventions to be used in conjunction with other therapeutics commonly employed in the treatment of diabetes and its complications.

Cancer and Risk of COVID-19 Through a General Community Survey.

Individuals with cancer may be at high risk for coronavirus disease 2019 (COVID-19) and adverse outcomes. However, evidence from large population-based studies examining whether cancer and cancer-related therapy exacerbates the risk of COVID-19 infection is still limited. Data were collected from the COVID Symptom Study smartphone application since March 29 through May 8, 2020. Among 23,266 participants with cancer and 1,784,293 without cancer, we documented 10,404 reports of a positive COVID-19 test. Compared with participants without cancer, those living with cancer had a 60% increased risk of a positive COVID-19 test. Among patients with cancer, current treatment with chemotherapy or immunotherapy was associated with a 2.2-fold increased risk of a positive test. The association between cancer and COVID-19 infection was stronger among participants >65 years and males. Future studies are needed to identify subgroups by tumor types and treatment regimens who are particularly at risk for COVID-19 infection and adverse outcomes.

Monoacylglycerol Acyltransferase 1 Knockdown Exacerbates Hepatic Ischemia/Reperfusion Injury in Mice With Hepatic Steatosis.

Nonalcoholic fatty liver disease (NAFLD) is becoming the most common indication for liver transplantation. The growing prevalence of NAFLD not only increases the demand for liver transplantation, but it also limits the supply of available organs because steatosis predisposes grafts to ischemia/reperfusion injury (IRI) and many steatotic grafts are discarded. We have shown that monoacylglycerol acyltransferase (MGAT) 1, an enzyme that converts monoacylglycerol to diacylglycerol, is highly induced in animal models and patients with NAFLD and is an important mediator in NAFLD-related insulin resistance. Herein, we sought to determine whether Mogat1 (the gene encoding MGAT1) knockdown in mice with hepatic steatosis would reduce liver injury and improve liver regeneration following experimental IRI. Antisense oligonucleotides (ASO) were used to knockdown the expression of Mogat1 in a mouse model of NAFLD. Mice then underwent surgery to induce IRI. We found that Mogat1 knockdown reduced hepatic triacylglycerol accumulation, but it unexpectedly exacerbated liver injury and mortality following experimental ischemia/reperfusion surgery in mice on a high-fat diet. The increased liver injury was associated with robust effects on the hepatic transcriptome following IRI including enhanced expression of proinflammatory cytokines and chemokines and suppression of enzymes involved in intermediary metabolism. These transcriptional changes were accompanied by increased signs of oxidative stress and an impaired regenerative response. We have shown that Mogat1 knockdown in a mouse model of NAFLD exacerbates IRI and inflammation and prolongs injury resolution, suggesting that Mogat1 may be necessary for liver regeneration following IRI and that targeting this metabolic enzyme will not be an effective treatment to reduce steatosis-associated graft dysfunction or failure.

Anxiety and depression symptoms after COVID-19 infection: results from the COVID Symptom Study app.

BACKGROUND: Mental health issues have been reported after SARS-CoV-2 infection. However, comparison to prevalence in uninfected individuals and contribution from common risk factors (eg, obesity and comorbidities) have not been examined. We identified how COVID-19 relates to mental health in the large community-based COVID Symptom Study. METHODS: We assessed anxiety and depression symptoms using two validated questionnaires in 413148 individuals between February and April 2021; 26998 had tested positive for SARS-CoV-2. We adjusted for physical and mental prepandemic comorbidities, body mass index (BMI), age and sex. FINDINGS: Overall, 26.4% of participants met screening criteria for general anxiety and depression. Anxiety and depression were slightly more prevalent in previously SARS-CoV-2-positive (30.4%) vs SARS-CoV-2-negative (26.1%) individuals. This association was small compared with the effect of an unhealthy BMI and the presence of other comorbidities, and not evident in younger participants (≤40 years). Findings were robust to multiple sensitivity analyses. Association between SARS-CoV-2 infection and anxiety and depression was stronger in individuals with recent (<30 days) versus more distant (>120 days) infection, suggesting a short-term effect. INTERPRETATION: A small association was identified between SARS-CoV-2 infection and anxiety and depression symptoms. The proportion meeting criteria for self-reported anxiety and depression disorders is only slightly higher than prepandemic.

Probable delirium is a presenting symptom of COVID-19 in frail, older adults: a cohort study of 322 hospitalised and 535 community-based older adults.

BACKGROUND: Frailty, increased vulnerability to physiological stressors, is associated with adverse outcomes. COVID-19 exhibits a more severe disease course in older, comorbid adults. Awareness of atypical presentations is critical to facilitate early identification. OBJECTIVE: To assess how frailty affects presenting COVID-19 symptoms in older adults. DESIGN: Observational cohort study of hospitalised older patients and self-report data for community-based older adults. SETTING: Admissions to St Thomas' Hospital, London with laboratory-confirmed COVID-19. Community-based data for older adults using the COVID Symptom Study mobile application. SUBJECTS: Hospital cohort: patients aged 65 and over (n = 322); unscheduled hospital admission between 1 March 2020 and 5 May 2020; COVID-19 confirmed by RT-PCR of nasopharyngeal swab. Community-based cohort: participants aged 65 and over enrolled in the COVID Symptom Study (n = 535); reported test-positive for COVID-19 from 24 March (application launch) to 8 May 2020. METHODS: Multivariable logistic regression analysis performed on age-matched samples from hospital and community-based cohorts to ascertain association of frailty with symptoms of confirmed COVID-19. RESULTS: Hospital cohort: significantly higher prevalence of probable delirium in the frail sample, with no difference in fever or cough. Community-based cohort: significantly higher prevalence of possible delirium in frailer, older adults and fatigue and shortness of breath. CONCLUSIONS: This is the first study demonstrating higher prevalence of probable delirium as a COVID-19 symptom in older adults with frailty compared to other older adults. This emphasises need for systematic frailty assessment and screening for delirium in acutely ill older patients in hospital and community settings. Clinicians should suspect COVID-19 in frail adults with delirium.

SNAP-25 phosphorylation at Ser187 is not involved in Ca2+ or phorbolester-dependent potentiation of synaptic release.

SNAP-25, one of the three SNARE-proteins responsible for synaptic release, can be phosphorylated by Protein Kinase C on Ser-187, close to the fusion pore. In neuroendocrine cells, this phosphorylation event potentiates vesicle recruitment into releasable pools, whereas the consequences of phosphorylation for synaptic release remain unclear. We mutated Ser-187 and expressed two mutants (S187C and S187E) in the context of the SNAP-25B-isoform in SNAP-25 knockout glutamatergic autaptic neurons. Whole-cell patch clamp recordings were performed to assess the effect of Ser-187 phosphorylation on synaptic transmission. Blocking phosphorylation by expressing the S187C mutant did not affect synapse density, basic evoked or spontaneous neurotransmission, the readily-releasable pool size or its Ca2+-independent or Ca2+-dependent replenishment. Furthermore, it did not affect the response to phorbol esters, which activate PKC. Expressing S187C in the context of the SNAP-25A isoform also did not affect synaptic transmission. Strikingly, the - potentially phosphomimetic - mutant S187E reduced spontaneous release and release probability, with the largest effect seen in the SNAP-25B isoform, showing that a negative charge in this position is detrimental for neurotransmission, in agreement with electrostatic fusion triggering. During the course of our experiments, we found that higher SNAP-25B expression levels led to decreased paired pulse potentiation, probably due to higher release probabilities. Under these conditions, the potentiation of evoked EPSCs by phorbol esters was followed by a persistent down-regulation, probably due to a ceiling effect. In conclusion, our results indicate that phosphorylation of Ser-187 in SNAP-25 is not involved in modulation of synaptic release by Ca2+ or phorbol esters.

Cardiovascular and Metabolic Effects of ANGPTL3 Antisense Oligonucleotides.

BACKGROUND: Epidemiologic and genomewide association studies have linked loss-of-function variants in ANGPTL3, encoding angiopoietin-like 3, with low levels of plasma lipoproteins. METHODS: We evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side-effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins. RESULTS: The treated mice had dose-dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low-density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple-dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, P<0.01 for all doses vs. placebo) and in levels of triglycerides (reductions of 33.2 to 63.1%), LDL cholesterol (1.3 to 32.9%), very-low-density lipoprotein cholesterol (27.9 to 60.0%), non-high-density lipoprotein cholesterol (10.0 to 36.6%), apolipoprotein B (3.4 to 25.7%), and apolipoprotein C-III (18.9 to 58.8%). Three participants who received the antisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events. CONCLUSIONS: Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in mice. Use of the same strategy to target human ANGPTL3 reduced levels of atherogenic lipoproteins in humans. (Funded by Ionis Pharmaceuticals; ClinicalTrials.gov number, NCT02709850 .).

Novel Reversible Model of Atherosclerosis and Regression Using Oligonucleotide Regulation of the LDL Receptor.

RATIONALE: Animal models have been used to explore factors that regulate atherosclerosis. More recently, they have been used to study the factors that promote loss of macrophages and reduction in lesion size after lowering of plasma cholesterol levels. However, current animal models of atherosclerosis regression require challenging surgeries, time-consuming breeding strategies, and methods that block liver lipoprotein secretion. OBJECTIVE: We sought to develop a more direct or time-effective method to create and then reverse hypercholesterolemia and atherosclerosis via transient knockdown of the hepatic LDLR (low-density lipoprotein receptor) followed by its rapid restoration. METHODS AND RESULTS: We used antisense oligonucleotides directed to LDLR mRNA to create hypercholesterolemia in wild-type C57BL/6 mice fed an atherogenic diet. This led to the development of lesions in the aortic root, aortic arch, and brachiocephalic artery. Use of a sense oligonucleotide replicating the targeted sequence region of the LDLR mRNA rapidly reduced circulating cholesterol levels because of recovery of hepatic LDLR expression. This led to a decrease in macrophages within the aortic root plaques and brachiocephalic artery, that is, regression of inflammatory cell content, after a period of 2 to 3 weeks. CONCLUSIONS: We have developed an inducible and reversible hepatic LDLR knockdown mouse model of atherosclerosis regression. Although cholesterol reduction decreased early en face lesions in the aortic arches, macrophage area was reduced in both early and late lesions within the aortic sinus after reversal of hypercholesterolemia. Our model circumvents many of the challenges associated with current mouse models of regression. The use of this technology will potentially expedite studies of atherosclerosis and regression without use of mice with genetic defects in lipid metabolism.

ApoC-III Glycoforms Are Differentially Cleared by Hepatic TRL (Triglyceride-Rich Lipoprotein) Receptors.

OBJECTIVE: ApoC-III (apolipoprotein C-III) glycosylation can predict cardiovascular disease risk. Higher abundance of disialylated (apoC-III2) over monosialylated (apoC-III1) glycoforms is associated with lower plasma triglyceride levels. Yet, it remains unclear whether apoC-III glycosylation impacts TRL (triglyceride-rich lipoprotein) clearance and whether apoC-III antisense therapy (volanesorsen) affects distribution of apoC-III glycoforms. Approach and Results: To measure the abundance of human apoC-III glycoforms in plasma over time, human TRLs were injected into wild-type mice and mice lacking hepatic TRL clearance receptors, namely HSPGs (heparan sulfate proteoglycans) or both LDLR (low-density lipoprotein receptor) and LRP1 (LDLR-related protein 1). ApoC-III was more rapidly cleared in the absence of HSPG (t1/2=25.4 minutes) than in wild-type animals (t1/2=55.1 minutes). In contrast, deficiency of LDLR and LRP1 (t1/2=56.1 minutes) did not affect clearance of apoC-III. After injection, a significant increase in the relative abundance of apoC-III2 was observed in HSPG-deficient mice, whereas the opposite was observed in mice lacking LDLR and LRP1. In patients, abundance of plasma apoC-III glycoforms was assessed after placebo or volanesorsen administration. Volanesorsen treatment correlated with a statistically significant 1.4-fold increase in the relative abundance of apoC-III2 and a 15% decrease in that of apoC-III1. The decrease in relative apoC-III1 abundance was strongly correlated with decreased plasma triglyceride levels in patients. CONCLUSIONS: Our results indicate that HSPGs preferentially clear apoC-III2. In contrast, apoC-III1 is more effectively cleared by LDLR/LRP1. Clinically, the increase in the apoC-III2/apoC-III1 ratio on antisense lowering of apoC-III might reflect faster clearance of apoC-III1 because this metabolic shift associates with improved triglyceride levels.

Genetic Deficiency of Flavin-Containing Monooxygenase 3 ( Fmo3) Protects Against Thrombosis but Has Only a Minor Effect on Plasma Lipid Levels-Brief Report.

Objective- FMO (flavin-containing monooxygenase) 3 converts bacterial-derived trimethylamine to trimethylamine N-oxide (TMAO), an independent risk factor for cardiovascular disease. We generated FMO3 knockout (FMO3KO) mouse to study its effects on plasma TMAO, lipids, glucose/insulin metabolism, thrombosis, and atherosclerosis. Approach and Results- Previous studies with an antisense oligonucleotide (ASO) knockdown strategy targeting FMO3 in LDLRKO (low-density lipoprotein receptor knockout) mice resulted in major reductions in TMAO levels and atherosclerosis, but also showed effects on plasma lipids, insulin, and glucose. Although FMO3KO mice generated via CRISPR/Cas9 technology bred onto the LDLRKO background did exhibit similar effects on TMAO levels, the effects on lipid metabolism were not as pronounced as with the ASO knockdown model. These differences could result from either off-target effects of the ASO or from a developmental adaptation to the FMO3 deficiency. To distinguish these possibilities, we treated wild-type and FMO3KO mice with control or FMO3 ASOs. FMO3-ASO treatment led to the same extent of lipid-lowering effects in the FMO3KO mice as the wild-type mice, indicating off-target effects. The levels of TMAO in LDLRKO mice fed an atherogenic diet are very low in both wild-type and FMO3KO mice, and no significant effect was observed on atherosclerosis. When FMO3KO and wild-type mice were maintained on a 0.5% choline diet, FMO3KO showed a marked reduction in both TMAO and in vivo thrombosis potential. Conclusions- FMO3KO markedly reduces systemic TMAO levels and thrombosis potential. However, the previously observed large effects of an FMO3 ASO on plasma lipid levels appear to be due partly to off-target effects.

Angiotensinogen and Megalin Interactions Contribute to Atherosclerosis-Brief Report.

Objective- AGT (Angiotensinogen) is the unique precursor of the renin-angiotensin system that is sequentially cleaved by renin and ACE (angiotensin-converting enzyme) to produce Ang II (angiotensin II). In this study, we determined how these renin-angiotensin components interact with megalin in kidney to promote atherosclerosis. Approach and Results- AGT, renin, ACE, and megalin were present in the renal proximal convoluted tubules of wild-type mice. Hepatocyte-specific AGT deficiency abolished AGT protein accumulation in proximal tubules and diminished Ang II concentrations in kidney, while renin was increased. Megalin was most abundant in kidney and exclusively present on the apical side of proximal tubules. Inhibition of megalin by antisense oligonucleotides (ASOs) led to ablation of AGT and renin proteins in proximal tubules, while leading to striking increases of urine AGT and renin concentrations, and 70% reduction of renal Ang II concentrations. However, plasma Ang II concentrations were unaffected. To determine whether AGT and megalin interaction contributes to atherosclerosis, we used both male and female low-density lipoprotein receptor-/- mice fed a saturated fat-enriched diet and administered vehicles (PBS or control ASO) or megalin ASO. Inhibition of megalin did not affect plasma cholesterol concentrations, but profoundly reduced atherosclerotic lesion size in both male and female mice. Conclusions- These results reveal a regulatory role of megalin in the intrarenal renin-angiotensin homeostasis and atherogenesis, positing renal Ang II to be an important contributor to atherosclerosis that is mediated through AGT and megalin interactions.

A review of available software for adaptive clinical trial design.

BACKGROUND/AIMS: The increasing cost of the drug development process has seen interest in the use of adaptive trial designs grow substantially. Accordingly, much research has been conducted to identify barriers to increasing the use of adaptive designs in practice. Several articles have argued that the availability of user-friendly software will be an important step in making adaptive designs easier to implement. Therefore, we present a review of the current state of software availability for adaptive trial design. METHODS: We review articles from 31 journals published in 2013-2017 that relate to methodology for adaptive trials to assess how often code and software for implementing novel adaptive designs is made available at the time of publication. We contrast our findings against these journals' policies on code distribution. We also search popular code repositories, such as Comprehensive R Archive Network and GitHub, to identify further existing user-contributed software for adaptive designs. From this, we are able to direct interested parties toward solutions for their problem of interest. RESULTS: Only 30% of included articles made their code available in some form. In many instances, articles published in journals that had mandatory requirements on code provision still did not make code available. There are several areas in which available software is currently limited or saturated. In particular, many packages are available to address group sequential design, but comparatively little code is present in the public domain to determine biomarker-guided adaptive designs. CONCLUSIONS: There is much room for improvement in the provision of software alongside adaptive design publications. In addition, while progress has been made, well-established software for various types of trial adaptation remains sparsely available.