Angiopoietin-like protein 3 (ANGPTL3) inhibits lipid clearance and is a promising target for managing cardiovascular disease. Here we investigated the effects of a high-sugar (high-fructose) diet on circulating ANGPTL3 concentrations in rhesus macaques. Plasma ANGPTL3 concentrations increased ~30% to 40% after 1 and 3 months of a high-fructose diet (both P < 0.001 vs. baseline). During fructose-induced metabolic dysregulation, plasma ANGPTL3 concentrations were positively correlated with circulating indices of insulin resistance [assessed with fasting insulin and the homeostatic model assessment of insulin resistance (HOMA-IR)], hypertriglyceridemia, adiposity (assessed as leptin), and systemic inflammation [C-reactive peptide (CRP)] and negatively correlated with plasma levels of the insulin-sensitizing hormone adropin. Multiple regression analyses identified a strong association between circulating APOC3 and ANGPTL3 concentrations. Higher baseline plasma levels of both ANGPTL3 and APOC3 were associated with an increased risk for fructose-induced insulin resistance. Fish oil previously shown to prevent insulin resistance and hypertriglyceridemia in this model prevented increases of ANGPTL3 without affecting systemic inflammation (increased plasma CRP and interleukin-6 concentrations). ANGPTL3 RNAi lowered plasma concentrations of ANGPTL3, triglycerides (TGs), VLDL-C, APOC3, and APOE. These decreases were consistent with a reduced risk of atherosclerosis. In summary, dietary sugar-induced increases of circulating ANGPTL3 concentrations after metabolic dysregulation correlated positively with leptin levels, HOMA-IR, and dyslipidemia. Targeting ANGPTL3 expression with RNAi inhibited dyslipidemia by lowering plasma TGs, VLDL-C, APOC3, and APOE levels in rhesus macaques.

Atherosclerosis-related CVD causes nearly 20 million deaths annually. Most patients are treated after plaques develop, so therapies must regress existing lesions. Current therapies reduce plaque volume, but targeting all apoB-containing lipoproteins with intensive combinations that include alirocumab or evinacumab, monoclonal antibodies against cholesterol-regulating proprotein convertase subtilisin/kexin type 9 and angiopoietin-like protein 3, may provide more benefit. We investigated the effect of such lipid-lowering interventions on atherosclerosis in APOE*3-Leiden.CETP mice, a well-established model for hyperlipidemia. Mice were fed a Western-type diet for 13 weeks and thereafter matched into a baseline group (euthanized at 13 weeks) and five groups that received diet alone (control) or with treatment [atorvastatin; atorvastatin and alirocumab; atorvastatin and evinacumab; or atorvastatin, alirocumab, and evinacumab (triple therapy)] for 25 weeks. We measured effects on cholesterol levels, plaque composition and morphology, monocyte adherence, and macrophage proliferation. All interventions reduced plasma total cholesterol (37% with atorvastatin to 80% with triple treatment; all P < 0.001). Triple treatment decreased non-HDL-C to 1.0 mmol/l (91% difference from control; P < 0.001). Atorvastatin reduced atherosclerosis progression by 28% versus control (P < 0.001); double treatment completely blocked progression and diminished lesion severity. Triple treatment regressed lesion size versus baseline in the thoracic aorta by 50% and the aortic root by 36% (both P < 0.05 vs. baseline), decreased macrophage accumulation through reduced proliferation, and abated lesion severity. Thus, high-intensive cholesterol-lowering triple treatment targeting all apoB-containing lipoproteins regresses atherosclerotic lesion area and improves lesion composition in mice, making it a promising potential approach for treating atherosclerosis.

Lipid droplets (LDs) are energy-storage organelles that are coated with hundreds of proteins, including members of the perilipin (PLIN) family. PLIN5 is highly expressed in oxidative tissues, including the liver, and is thought to play a key role in uncoupling LD accumulation from lipotoxicity; however, the mechanisms behind this action are incompletely defined. We investigated the role of hepatic PLIN5 in inflammation and lipotoxicity in a murine model under both fasting and refeeding conditions and in hepatocyte cultures. PLIN5 ablation with antisense oligonucleotides triggered a pro-inflammatory response in livers from mice only under fasting conditions. Similarly, PLIN5 mitigated lipopolysaccharide- or palmitic acid-induced inflammatory responses in hepatocytes. During fasting, PLIN5 was also required for the induction of autophagy, which contributed to its anti-inflammatory effects. The ability of PLIN5 to promote autophagy and prevent inflammation were dependent upon signaling through sirtuin 1 (SIRT1), which is known to be activated in response to nuclear PLIN5 under fasting conditions. Taken together, these data show that PLIN5 signals via SIRT1 to promote autophagy and prevent FA-induced inflammation as a means to maintain hepatocyte homeostasis during periods of fasting and FA mobilization.

apoB exists as apoB100 and apoB48, which are mainly found in hepatic VLDLs and intestinal chylomicrons, respectively. Elevated plasma levels of apoB-containing lipoproteins (Blps) contribute to coronary artery disease, diabetes, and other cardiometabolic conditions. Studying the mechanisms that drive the assembly, intracellular trafficking, secretion, and function of Blps remains challenging. Our understanding of the intracellular and intraorganism trafficking of Blps can be greatly enhanced, however, with the availability of fusion proteins that can help visualize Blp transport within cells and between tissues. We designed three plasmids expressing human apoB fluorescent fusion proteins: apoB48-GFP, apoB100-GFP, and apoB48-mCherry. In Cos-7 cells, transiently expressed fluorescent apoB proteins colocalized with calnexin and were only secreted if cells were cotransfected with microsomal triglyceride transfer protein. The secreted apoB-fusion proteins retained the fluorescent protein and were secreted as lipoproteins with flotation densities similar to plasma HDL and LDL. In a rat hepatoma McA-RH7777 cell line, the human apoB100 fusion protein was secreted as VLDL- and LDL-sized particles, and the apoB48 fusion proteins were secreted as LDL- and HDL-sized particles. To monitor lipoprotein trafficking in vivo, the apoB48-mCherry construct was transiently expressed in zebrafish larvae and was detected throughout the liver. These experiments show that the addition of fluorescent proteins to the C terminus of apoB does not disrupt their assembly, localization, secretion, or endocytosis. The availability of fluorescently labeled apoB proteins will facilitate the exploration of the assembly, degradation, and transport of Blps and help to identify novel compounds that interfere with these processes via high-throughput screening.

The hydrolysis of triglycerides in triglyceride-rich lipoproteins by LPL is critical for the delivery of triglyceride-derived fatty acids to tissues, including heart, skeletal muscle, and adipose tissues. Physiologically active LPL is normally bound to the endothelial cell protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), which transports LPL across endothelial cells, anchors LPL to the vascular wall, and stabilizes LPL activity. Disruption of LPL-GPIHBP1 binding significantly alters triglyceride metabolism and lipid partitioning. In this study, we modified the NanoLuc® Binary Technology split-luciferase system to develop a novel assay that monitors the binding of LPL to GPIHBP1 on endothelial cells in real time. We validated the specificity and sensitivity of the assay using endothelial lipase and a mutant version of LPL and found that this assay reliably and specifically detected the interaction between LPL and GPIHBP1. We then interrogated various endogenous and exogenous inhibitors of LPL-mediated lipolysis for their ability to disrupt the binding of LPL to GPIHBP1. We found that angiopoietin-like (ANGPTL)4 and ANGPTL3-ANGPTL8 complexes disrupted the interactions of LPL and GPIHBP1, whereas the exogenous LPL blockers we tested (tyloxapol, poloxamer-407, and tetrahydrolipstatin) did not. We also found that chylomicrons could dissociate LPL from GPIHBP1 and found evidence that this dissociation was mediated in part by the fatty acids produced by lipolysis. These results demonstrate the ability of this assay to monitor LPL-GPIHBP1 binding and to probe how various agents influence this important complex.

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (^–/–) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R^–/–) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by ²H/¹³C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

The pregnane X receptor (PXR) is a nuclear receptor that can be activated by numerous drugs and xenobiotic chemicals. PXR thereby functions as a xenobiotic sensor to coordinately regulate host responses to xenobiotics by transcriptionally regulating many genes involved in xenobiotic metabolism. We have previously reported that PXR has pro-atherogenic effects in animal models, but how PXR contributes to atherosclerosis development in different tissues or cell types remains elusive. In this study, we generated an LDL receptor-deficient mouse model with myeloid-specific PXR deficiency (PXR^MyeLDLR^–/–) to elucidate the role of macrophage PXR signaling in atherogenesis. The myeloid PXR deficiency did not affect metabolic phenotypes and plasma lipid profiles, but PXR^MyeLDLR^–/– mice had significantly decreased atherosclerosis at both aortic root and brachiocephalic arteries compared with control littermates. Interestingly, the PXR deletion did not affect macrophage adhesion and migration properties, but reduced lipid accumulation and foam cell formation in the macrophages. PXR deficiency also led to decreased expression of the scavenger receptor CD36 and impaired lipid uptake in macrophages of the PXR^MyeLDLR^–/– mice. Further, RNA-Seq analysis indicated that treatment with a prototypical PXR ligand affects the expression of many atherosclerosis-related genes in macrophages in vitro. These findings reveal a pivotal role of myeloid PXR signaling in atherosclerosis development and suggest that PXR may be a potential therapeutic target in atherosclerosis management.

Activation of microglia and astrocytes secondary to inflammatory processes contributes to the development and perpetuation of pain with a neuropathic phenotype. This pain state presents as a chronic debilitating condition and affects a large population of patients with conditions like rheumatoid arthritis and diabetes, or after surgery, trauma, or chemotherapy. Here, we review the regulation of lipid rafts in glial cells and the role they play as a key component of neuroinflammatory sensitization of central pain signaling pathways. In this context, we introduce the concept of an inflammaraft (i-raft), enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as an organizing platform to initiate inflammatory signaling and the cellular response. Characteristics of the inflammaraft include increased relative abundance of lipid rafts in inflammatory cells, increased content of cholesterol per raft, and increased levels of inflammatory receptors, such as toll-like receptor (TLR)4, adaptor molecules, ion channels, and enzymes in lipid rafts. This inflammaraft motif serves an important role in the membrane assembly of protein complexes, for example, TLR4 dimerization. Operating within this framework, we demonstrate the involvement of inflammatory receptors, redox molecules, and ion channels in the inflammaraft formation and the regulation of cholesterol and sphingolipid metabolism in the inflammaraft maintenance and disruption. Strategies for targeting inflammarafts, without affecting the integrity of lipid rafts in noninflammatory cells, may lead to developing novel therapies for neuropathic pain states and other neuroinflammatory conditions.

Cholesterol/sphingolipid-rich membrane domains, known as lipid rafts or membrane rafts, play a critical role in the compartmentalization of signaling pathways. Physical segregation of proteins in lipid rafts may modulate the accessibility of proteins to regulatory or effector molecules. Thus, lipid rafts serve as sorting platforms and hubs for signal transduction proteins. Cancer cells contain higher levels of intracellular cholesterol and lipid rafts than their normal non-tumorigenic counterparts. Many signal transduction processes involved in cancer development (insulin-like growth factor system and phosphatidylinositol 3-kinase-AKT) and metastasis [cluster of differentiation (CD)44] are dependent on or modulated by lipid rafts. Additional proteins playing an important role in several malignant cancers (e.g., transmembrane glycoprotein mucin 1) are also being detected in association with lipid rafts, suggesting a major role of lipid rafts in tumor progression. Conversely, lipid rafts also serve as scaffolds for the recruitment and clustering of Fas/CD95 death receptors and downstream signaling molecules leading to cell death-promoting raft platforms. The partition of death receptors and downstream signaling molecules in aggregated lipid rafts has led to the formation of the so-called cluster of apoptotic signaling molecule-enriched rafts, or CASMER, which leads to apoptosis amplification and can be pharmacologically modulated. These death-promoting rafts can be viewed as a linchpin from which apoptotic signals are launched. In this review, we discuss the involvement of lipid rafts in major signaling processes in cancer cells, including cell survival, cell death, and metastasis, and we consider the potential of lipid raft modulation as a promising target in cancer therapy.

The estimation results might be incorrect in PROC PANEL when the RANONE and NOINT options are specified in the MODEL statement.

When you query a PI tag name or element that contains a special character, such as an ampersand (&), you might receive the following error:

When you run an SQL query using SAS/ACCESS Interface to ODBC under the following conditions, you might receive an error: You run SAS 9.4M5 (TS1M5) or SAS 9.4M6 (TS1M6)  i

Large numeric values consisting of 16 digits in SAS might be incorrect when written to Hadoop for columns defined with the BIGINT data type.  This problem was introduced in SAS 9

When you register an Oracle table to the metadata, it might fail and generate an error similar to the following: "ERROR: An exception has been encountered...ERROR: Read Access Violation METALIB..."

If a bulk-loading process fails when you use SAS with SAS/ACCESS Interface to Oracle, you will receive the warning: "WARNING: All or some rows were rejected/discarded.: The actual error is "SQL*Loader-2026: The load was aborted because SQL

The METADATA procedure might return an error similar to one of the following: