In SAS Customer Intelligence Studio, the following error is displayed when you update a new Link  node in a diagram:   imgalt="Link: MAIQService:executeFastPath:" src="{fusion_665

In SAS Business Rules Manager 3.3, the initial loading of a rule set and a rule flow takes significantly longer than it does in release 3.2. When this problem happens, long time gaps are evident in the local

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An error might occur when you use the SAS 9 BULKLOAD= and BULKEXTRACT= options load data to or extract data from HP Neoview on the HP Itanium platform. The problem occurs because Hewlett-Packard changed the name of one of

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When viewing a SAS Visual Analytics report, you might intermittently see an error that includes content similar to the following:

When you use SAS/ACCESS Interface to PostgreSQL to query a Yellowbrick database, the SAS OBS= option is not generating a limit clause on the query that is passed to the database. Click the

In SAS Infrastructure for Risk Management, the message "ERROR: Could not move and link one or more files to..." occurs while running a job-flow instance if an orphaned folder exists in the persistent area.

The SAS 9.4M4 (TS1M4) Hot Fix D7G004 for ODS Templates installs national language support (NLS) content regardless of whether the languages were installed during the initial deployment. Having sparse

SAS Web Report Studio enables you to link reports based on a group break value. However, when you click the link, it might fail with an HTTP 400 error. The exact message you see depends on which browser you are u

When you log on to the SAS Risk Governance Framework and choose a solution, the web application might fail to load the solution content. When the problem occurs, you continue to see "Loading..." on the screen, an

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism by inducing the degradation of hepatic low-density lipoprotein receptor (LDLR). Plasma PCSK9 has two main molecular forms: a 62-kDa mature form (PCSK9_62) and a 55-kDa, furin-cleaved form (PCSK9_55). PCSK9_55 is considered less active than PCSK9_62 in degrading LDLR. We aimed to identify the site of PCSK9_55 formation (intra- vs. extracellular) and to further characterize the LDLR-degradative function of PCSK9_55 relative to PCSK9_62. Co-expressing PCSK9_62 with furin in cell culture induced formation of PCSK9_55, most of which was found in the extracellular space. Under the same conditions we found that: i) adding a cell-permeable furin inhibitor preferentially decreased the formation of PCSK9_55 extracellularly; ii) using pulse-chase, we observed the formation of PCSK9_55 exclusively extracellularly in a time-dependent manner. A recombinant form of PCSK9_55 was efficiently produced but displayed impaired secretion that resulted in its intracellular trapping. However, the non-secreted PCSK9_55 was able to induce degradation of LDLR, though with 50% lower efficiency compared with PCSK9_62. Collectively, our data show that PCSK9_55 is generated in the extracellular space, and that intracellular PCSK9_55 is not secreted but retains the ability to degrade the LDLR through an intracellular pathway.

Smith-Lemli-Opitz Syndrome (SLOS) is a developmental disorder (OMIM #270400) caused by autosomal recessive mutations in the Dhcr7 gene, which encodes the enzyme 3β-hydroxysterol-7 reductase. SLOS patients present clinically with dysmorphology and neurological, behavioral and cognitive defects, with characteristically elevated levels of 7-dehydrocholesterol (7-DHC) in all bodily tissues and fluids. Previous mouse models of SLOS have been hampered by postnatal lethality when Dhcr7 is knocked out globally, while a hypomorphic mouse model showed improvement in the biochemical phenotype with ageing, and did not manifest most other characteristic features of SLOS. We report the generation of a conditional knockout of Dhcr7 (Dhcr7flx/flx), validated by generating a mouse with a liver-specific deletion (Dhcr7L-KO). Phenotypic characterization of liver-specific knockout mice revealed no significant changes in viability, fertility, growth curves, liver architecture, hepatic triglyceride secretion, or parameters of systemic glucose homeostasis. Furthermore, qPCR and RNA-Seq analyses of livers revealed no perturbations in pathways responsible for cholesterol synthesis, either in male or female Dhcr7L-KO mice, suggesting hepatic disruption of post-squalene cholesterol synthesis leads to minimal impact on sterol metabolism in the liver. This validated conditional Dhcr7 knockout model may now allow us to systematically explore the pathophysiology of SLOS, by allowing for temporal, cell and tissue-specific loss of DHCR7.

Bacterial lipopolysaccharides (LPSs or endotoxins) can bind most proteins of the lipid transfer/LPS-binding protein (LT/LBP) family in host organisms. The LPS-bound LT/LBP proteins then trigger either an LPS-induced proinflammatory cascade or LPS binding to lipoproteins that are involved in endotoxin inactivation and detoxification. Cholesteryl ester transfer protein (CETP) is an LT/LBP member, but its impact on LPS metabolism and sepsis outcome is unclear. Here, we performed fluorescent LPS transfer assays to assess the ability of CETP to bind and transfer LPS. The effects of intravenous (iv) infusion of purified LPS or polymicrobial infection (cecal ligation and puncture [CLP]) were compared in transgenic mice expressing human CETP and wild-type mice naturally having no CETP activity. CETP displayed no LPS transfer activity in vitro, but it tended to reduce biliary excretion of LPS in vivo. The CETP expression in mice was associated with significantly lower basal plasma lipid levels and with higher mortality rates in both models of endotoxemia and sepsis. Furthermore, CETPTg plasma modified cytokine production of macrophages in vitro. In conclusion, despite having no direct LPS binding and transfer property, human CETP worsens sepsis outcomes in mice by altering the protective effects of plasma lipoproteins against endotoxemia, inflammation, and infection.

Apolipoproteins C-I, C-II and C-III interact with ApoE to regulate lipoprotein metabolism and contribute to Alzheimer’s disease pathophysiology. In plasma, apoC-I and C-II exist as truncated isoforms, while apoC-III exhibits multiple glycoforms. This study aimed to 1. delineate apoC-I, C-II and C-III isoform profiles in CSF and plasma in a cohort of non-demented older individuals (n = 61), and 2. examine the effect of APOE4 on these isoforms and their correlation with CSF Aβ42, a surrogate of brain amyloid accumulation. The isoforms of the apoCs were immunoaffinity enriched and measured with MALDI-TOF mass spectrometry, revealing a significantly higher percentage of truncated apoC-I and apoC-II in CSF compared to matched plasma, with positive correlation between CSF and plasma. A greater percentage of monosialylated and disialylated apoC-III isoforms was detected in CSF, accompanied by a lower percentage of the two non-sialylated apoC-III isoforms, with significant linear correlations between CSF and plasma. Furthermore, a greater percentage of truncated apoC-I in CSF, and apoC-II in plasma and CSF, was observed in individuals carrying at least one apoE E4 allele. Increased apoC-I and apoC-II truncations were  associated with lower CSF Aβ42. Finally, monosialylated apoC-III was lower, and disialylated apoC-III greater in the CSF of E4 carriers. Together, these results reveal distinct patterns of the apoCs isoforms in CSF, implying CSF-specific apoCs processing. These patterns were accentuated in APOE E4 allele carriers, suggesting an association between APOE4 genotype and Alzheimer’s disease pathology with apoCs processing and function in the brain.

Perilipin (PLIN) 5 is a lipid droplet-associated protein that coordinates intracellular lipolysis in highly oxidative tissues and is thought to regulate lipid metabolism in response to phosphorylation by protein kinase A (PKA). We sought to identify PKA phosphorylation sites in PLIN5 and assess their functional relevance in cultured cells and the livers of mice. We detected phosphorylation on S155, S161 and S163 of recombinant PLIN5 by PKA in vitro and identified S155 as a functionally important site for lipid metabolism. Expression of phosphorylation-defective PLIN5 S155A in Plin5 null cells resulted in decreased rates of lipolysis and triglyceride-derived fatty acid oxidation compared with cells expressing wildtype PLIN5. These differences in lipid metabolism were not associated with differences in the cellular distribution of PLIN5. Rather, FLIM-FRET analysis of protein-protein interactions showed that PLIN5 S155 phosphorylation regulates PLIN5 interaction with adipose triglyceride lipase (ATGL) at the lipid droplet, but not with the co-activator of ATGL, α-β hydrolase domain-containing 5 (ABHD5). Re-expression of PLIN5 S155A in the liver of Plin5 liver-specific null mice reduced lipolysis when compared to mice with wildtype PLIN5 re-expression, but was not associated with other changes in hepatic lipid metabolism, such as fatty acid oxidation, de novo lipogenesis and triglyceride secretion. Furthermore, glycemic control was impaired in mice with expression of PLIN5 S155A compared with mice expressing PLIN5. Together, these studies demonstrate that PLIN5 S155 is required for PKA-mediated lipolysis and builds on the body of evidence demonstrating a critical role for PLIN5 in coordinating lipid and glucose metabolism

Genome-wide association studies (GWAS) have implicated ~380 genetic loci for plasma lipid regulation. However, these loci only explain 17-27% of the trait variance and a comprehensive understanding of the molecular mechanisms has not been achieved. In this study, we utilized an integrative genomics approach leveraging diverse genomic data from human populations to investigate whether genetic variants associated with various plasma lipid traits, namely total cholesterol (TC), high and low density lipoprotein cholesterol (HDL and LDL), and triglycerides (TG), from GWAS were concentrated on specific parts of tissue-specific gene regulatory networks. In addition to the expected lipid metabolism pathways, gene subnetworks involved in ‘interferon signaling’, ‘autoimmune/immune activation’, ‘visual transduction’, and ‘protein catabolism’ were significantly associated with all lipid traits. Additionally, we detected trait-specific subnetworks, including cadherin-associated subnetworks for LDL, glutathione metabolism for HDL, valine, leucine and isoleucine biosynthesis for TC, and insulin signaling and complement pathways for TG. Finally, utilizing gene-gene relations revealed by tissue-specific gene regulatory networks, we detected both known (e.g. APOH, APOA4, and ABCA1) and novel (e.g. F2 in adipose tissue) key regulator genes in these lipid-associated subnetworks. Knockdown of the F2 gene (Coagulation Factor II, Thrombin) in 3T3-L1 and C3H10T1/2 adipocytes reduced gene expression of Abcb11, Apoa5, Apof, Fabp1, Lipc, and Cd36, reduced intracellular adipocyte lipid content, and increased extracellular lipid content, supporting a link between adipose thrombin and lipid regulation. Our results shed light on the complex mechanisms underlying lipid metabolism and highlight potential novel targets for lipid regulation and lipid-associated diseases.

Angiopoietin-like protein (ANGPTL)3 regulates plasma lipids by inhibiting LPL and endothelial lipase (EL). ANGPTL3 inactivation lowers LDL-C independently of the classical LDLR-mediated pathway and represents a promising therapeutic approach for individuals with homozygous familial hypercholesterolemia due to LDLR mutations. Yet, how ANGPTL3 regulates LDL-C levels is unknown. Here, we demonstrate in hyperlipidemic humans and mice that ANGPTL3 controls VLDL catabolism upstream of LDL. Using kinetic, lipidomic, and biophysical studies, we show that ANGPTL3 inhibition reduces VLDL-lipid content and size, generating remnant particles that are efficiently removed from the circulation. This suggests that ANGPTL3 inhibition lowers LDL-C by limiting LDL particle production. Mechanistically, we discovered that EL is a key mediator of ANGPTL3’s novel pathway. Our experiments revealed that, although dispensable in the presence of LDLR, EL-mediated processing of VLDL becomes critical for LDLR-independent particle clearance. In the absence of EL and LDLR, ANGPTL3 inhibition perturbed VLDL catabolism, promoted accumulation of atypical remnants, and failed to reduce LDL-C. Taken together, we uncover ANGPTL3 at the helm of a novel EL-dependent pathway that lowers LDL-C in the absence of LDLR.

Lipoprotein (a) [Lp(a)] is a risk factor for CVD and a target of therapy, but Lp(a) measurements are not globally standardized. Commercially available assays generally use polyclonal antibodies that detect multiple sites within the kringle (K)IV₂ repeat region of Lp(a) and may lead to inaccurate assessments of plasma levels. With increasing awareness of Lp(a) as a cardiovascular risk factor and the active clinical development of new potential therapeutic approaches, the broad availability of reagents capable of providing isoform independence of Lp(a) measurements is paramount. To address this issue, we generated a murine monoclonal antibody that binds to only one site on apo(a). A BALB/C mouse was immunized with a truncated version of apo(a) that contained eight total KIV repeats, including only one copy of KIV₂. We generated hybridomas, screened them, and successfully produced a KIV₂-independent monoclonal antibody, named LPA-KIV9. Using a variety of truncated apo(a) constructs to map its binding site, we found that LPA-KIV9 binds to KIV₉ without binding to plasminogen. Fine peptide mapping revealed that LPA-KIV9 bound to the sequence ⁴⁰⁷⁶LETPTVV⁴⁰⁸² on KIV₉. In conclusion, the generation of monoclonal antibody LPA-KIV9 may be a useful reagent in basic research studies and in the clinical application of Lp(a) measurements.

TG-rich lipoprotein (TRL)-related biomarkers, including TRL-cholesterol (TRL-C), remnant-like lipoprotein particle-cholesterol (RLP-C), and apoC-III have been associated with atherosclerosis. However, their prognostic values have not been fully determined, especially in patients with previous CAD. This study aimed to examine the associations of TRL-C, RLP-C, and apoC-III with incident cardiovascular events (CVEs) in the setting of secondary prevention of CAD. Plasma TRL-C, RLP-C, and total apoC-III were directly measured. A total of 4,355 participants with angiographically confirmed CAD were followed up for the occurrence of CVEs. During a median follow-up period of 5.1 years (interquartile range: 3.9–6.4 years), 543 (12.5%) events occurred. Patients with incident CVEs had significantly higher levels of TRL-C, RLP-C, and apoC-III than those without events. Multivariable Cox analysis indicated that a log unit increase in TRL-C, RLP-C, and apoC-III increased the risk of CVEs by 49% (95% CI: 1.16–1.93), 21% (95% CI: 1.09–1.35), and 40% (95% CI: 1.11–1.77), respectively. High TRL-C, RLP-C, and apoC-III were also independent predictors of CVEs in individuals with LDL-C levels ≤1.8 mmol/l (n = 1,068). The addition of RLP-C level to a prediction model resulted in a significant increase in discrimination, and all three TRL biomarkers improved risk reclassification. Thus, TRL-C, RLP-C, and apoC-III levels were independently associated with incident CVEs in Chinese CAD patients undergoing statin therapy.

Sphingolipids have become established participants in the pathogenesis of obesity and its associated maladies. Sphingosine kinase 1 (SPHK1), which generates S1P, has been shown to increase in liver and adipose of obese humans and mice and to regulate inflammation in hepatocytes and adipose tissue, insulin resistance, and systemic inflammation in mouse models of obesity. Previous studies by us and others have demonstrated that global sphingosine kinase 1 KO mice are protected from diet-induced obesity, insulin resistance, systemic inflammation, and NAFLD, suggesting that SPHK1 may mediate pathological outcomes of obesity. As adipose tissue dysfunction has gained recognition as a central instigator of obesity-induced metabolic disease, we hypothesized that SPHK1 intrinsic to adipocytes may contribute to HFD-induced metabolic pathology. To test this, we depleted Sphk1 from adipocytes in mice (SK1^fatKO) and placed them on a HFD. In contrast to our initial hypothesis, SK1^fatKO mice displayed greater weight gain on HFD and exacerbated impairment in glucose clearance. Pro-inflammatory cytokines and neutrophil content of adipose tissue were similar, as were levels of circulating leptin and adiponectin. However, SPHK1-null adipocytes were hypertrophied and had lower basal lipolytic activity. Interestingly, hepatocyte triacylglycerol accumulation and expression of pro-inflammatory cytokines and collagen 1a1 were exacerbated in SK1^fatKO mice on a HFD, implicating a specific role for adipocyte SPHK1 in adipocyte function and inter-organ cross-talk that maintains overall metabolic homeostasis in obesity. Thus, SPHK1 serves a previously unidentified essential homeostatic role in adipocytes that protects from obesity-associated pathology. These findings may have implications for pharmacological targeting of the SPHK1/S1P signaling axis.

Lipoprotein (a) [Lp(a)] is a well-known risk factor for cardiovascular disease, but analysis on Lp(a) and renal dysfunction is scarce. We aimed to investigate prospectively the association of serum Lp(a) with the risk of reduced renal function, and further investigated whether diabetic or hypertensive status modified such association. Six thousand two hundred and fifty-seven Chinese adults aged ≤40 years and free of reduced renal function at baseline were included in the study. Reduced renal function was defined as estimated glomerular filtration rate <60 ml/min/1.73 m². During a mean follow-up of 4.4 years, 158 participants developed reduced renal function. Each one-unit increase in log₁₀-Lp(a) (milligrams per deciliter) was associated with a 1.99-fold (95% CI 1.15–3.43) increased risk of incident reduced renal function; the multivariable-adjusted odds ratio (OR) for the highest tertile of Lp(a) was 1.61 (95% CI 1.03–2.52) compared with the lowest tertile (P for trend = 0.03). The stratified analysis showed the association of serum Lp(a) and incident reduced renal function was more prominent in participants with prevalent diabetes [OR 4.04, 95% CI (1.42–11.54)] or hypertension [OR 2.18, 95% CI (1.22–3.89)]. A stronger association was observed in the group with diabetes and high Lp(a) (>25 mg/dl), indicating a combined effect of diabetes and high Lp(a) on the reduced renal function risk. An elevated Lp(a) level was independently associated with risk of incident reduced renal function, especially in diabetic or hypertensive patients.

Cognitive decline with age is a harmful process that can reduce quality of life. Multiple factors have been established to contribute to cognitive decline, but the overall etiology remains unknown. Here, we hypothesized that cognitive dysfunction is mediated, in part, by increased levels of inflammatory cytokines that alter allopregnanolone (AlloP) levels, an important neurosteroid in the brain. We assessed the levels and regulation of AlloP and the effects of AlloP supplementation on cognitive function in 4-month-old and 24-month-old male C57BL/6 mice. With age, the expression of enzymes involved in the AlloP synthetic pathway was decreased and corticosterone (CORT) synthesis increased. Supplementation of AlloP improved cognitive function. Interestingly, interleukin 6 (IL-6) infusion in young animals significantly reduced the production of AlloP compared with controls. It is notable that inhibition of IL-6 with its natural inhibitor, soluble membrane glycoprotein 130, significantly improved spatial memory in aged mice. These findings were supported by in vitro experiments in primary murine astrocyte cultures, indicating that IL-6 decreases production of AlloP and increases CORT levels. Our results indicate that age-related increases in IL-6 levels reduce progesterone substrate availability, resulting in a decline in AlloP levels and an increase in CORT. Furthermore, our results indicate that AlloP is a critical link between inflammatory cytokines and the age-related decline in cognitive function.

Accompanied with nutrition transition, non-HDL-C levels of individuals in Asian countries has increased rapidly, which has caused the global epicenter of nonoptimal cholesterol to shift from Western countries to Asian countries. Thus, it is critical to underline major genetic and dietary determinants. In the current study of 2,330 Chinese individuals, genetic risk scores (GRSs) were calculated for total cholesterol (TC; GRS_TC, 57 SNPs), LDL-C (GRS_LDL-C, 45 SNPs), and HDL-C (GRS_HDL-C, 65 SNPs) based on SNPs from the Global Lipid Genetics Consortium study. Cholesterol intake was estimated by a 74-item food-frequency questionnaire. Associations of dietary cholesterol intake with plasma TC and LDL-C strengthened across quartiles of the GRS_TC (effect sizes: –0.29, 0.34, 2.45, and 6.47; P_interaction = 0.002) and GRS_LDL-C (effect sizes: –1.35, 0.17, 5.45, and 6.07; P_interaction = 0.001), respectively. Similar interactions with non-HDL-C were observed between dietary cholesterol and GRS_TC (P_interaction = 0.001) and GRS_LDL-C (P_interaction = 0.004). The adverse effects of GRS_TC on TC (effect sizes across dietary cholesterol quartiles: 0.51, 0.82, 1.21, and 1.31; P_interaction = 0.023) and GRS_LDL-C on LDL-C (effect sizes across dietary cholesterol quartiles: 0.66, 0.52, 1.12, and 1.56; P_interaction = 0.020) were more profound in those having higher cholesterol intake compared with those with lower intake. Our findings suggest significant interactions between genetic susceptibility and dietary cholesterol intake on plasma cholesterol profiles in a Chinese population.

Nonsmall cell lung cancer (NSCLC) is a leading cause of cancer-related deaths. While mutations in Kras and overexpression of Myc are commonly found in patients, the role of altered lipid metabolism in lung cancer and its interplay with oncogenic Myc is poorly understood. Here we use a transgenic mouse model of Kras-driven lung adenocarcinoma with reversible activation of Myc combined with surface analysis lipid profiling of lung tumors and transcriptomics to study the effect of Myc activity on cholesterol homeostasis. Our findings reveal that the activation of Myc leads to the accumulation of cholesteryl esters (CEs) stored in lipid droplets. Subsequent Myc deactivation leads to further increases in CEs, in contrast to tumors in which Myc was never activated. Gene expression analysis linked cholesterol transport and storage pathways to Myc activity. Our results suggest that increased Myc activity is associated with increased cholesterol influx, reduced efflux, and accumulation of CE-rich lipid droplets in lung tumors. Targeting cholesterol homeostasis is proposed as a promising avenue to explore for novel treatments of lung cancer, with diagnostic and stratification potential in human NSCLC.

Familial LCAT deficiency (FLD) is a rare genetic disorder of HDL metabolism, caused by loss-of-function mutations in the LCAT gene and characterized by a variety of symptoms including corneal opacities and kidney failure. Renal disease represents the leading cause of morbidity and mortality in FLD cases. However, the prognosis is not known and the rate of deterioration of kidney function is variable and unpredictable from patient to patient. In this article, we present data from a follow-up of the large Italian cohort of FLD patients, who have been followed for an average of 12 years. We show that renal failure occurs at the median age of 46 years, with a median time to a second recurrence of 10 years. Additionally, we identify high plasma unesterified cholesterol level as a predicting factor for rapid deterioration of kidney function. In conclusion, this study highlights the severe consequences of FLD, underlines the need of correct early diagnosis and referral of patients to specialized centers, and highlights the urgency for effective treatments to prevent or slow renal disease in patients with LCAT deficiency.

The dysregulation of myeloid-derived cell metabolism can drive atherosclerosis. AMP-activated protein kinase (AMPK) controls various aspects of macrophage dynamics and lipid homeostasis, which are important during atherogenesis. Using LysM-Cre to drive the deletion of both the α1 and α2 catalytic subunits (MacKO), we aimed to clarify the role of myeloid-specific AMPK signaling in male and female mice made acutely atherosclerotic by injection of AAV vector encoding a gain-of-function mutant PCSK9 (PCSK9-AAV) and WD feeding. After 6 weeks of WD feeding, mice received a daily injection of either the AMPK activator A-769662 or a vehicle control for an additional 6 weeks. Following this (12 weeks total), we assessed myeloid cell populations and differences between genotype or sex were not observed. Similarly, aortic sinus plaque size, lipid staining, and necrotic area did not differ in male and female MacKO mice compared with their littermate floxed controls. Moreover, therapeutic intervention with A-769662 showed no treatment effect. There were also no observable differences in the amount of circulating total cholesterol or triglyceride, and only minor differences in the levels of inflammatory cytokines between groups. Finally, CD68+ area and markers of autophagy showed no effect of either lacking AMPK signaling or AMPK activation. Our data suggest that while defined roles for each catalytic AMPK subunit have been identified, complete deletion of myeloid AMPK signaling does not significantly impact atherosclerosis. Additionally, these findings suggest that intervention with the first-generation AMPK activator A-769662 is not able to stem the progression of atherosclerosis.

Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apo(a). The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analog, -aminocaproic acid. Interestingly, KIV-2 presents a lysine binding site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in surface plasmon resonance experiments; and with the LBS being nonfunctional, we propose to rename it "pseudo-LBS". Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a)-related diseases and to facilitate the design of specific therapeutic drugs.

The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin’s anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp^–/–) mice and age-weight matched wild-type control mice. Young (10–12-week-old) L-Mttp^–/– mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp^–/– mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKC activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp^–/– mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKC activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp^–/– mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKC activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp^–/– mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp^–/– mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp^–/– mice.

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

The adipocyte-derived hormone leptin increases trafficking of KATP and Kv2.1 channels to the pancreatic β-cell surface, resulting in membrane hyperpolarization and suppression of insulin secretion. We have previously shown that this effect of leptin is mediated by the NMDA subtype of glutamate receptors (NMDARs). It does so by potentiating NMDAR activity, thus enhancing Ca2+ influx and the ensuing downstream signaling events that drive channel trafficking to the cell surface. However, the molecular mechanism by which leptin potentiates NMDARs in β-cells remains unknown. Here, we report that leptin augments NMDAR function via Src kinase–mediated phosphorylation of the GluN2A subunit. Leptin-induced membrane hyperpolarization diminished upon pharmacological inhibition of GluN2A but not GluN2B, indicating involvement of GluN2A-containing NMDARs. GluN2A harbors tyrosine residues that, when phosphorylated by Src family kinases, potentiate NMDAR activity. We found that leptin increases phosphorylation of Tyr-418 in Src, an indicator of kinase activation. Pharmacological inhibition of Src or overexpression of a kinase-dead Src mutant prevented the effect of leptin, whereas a Src kinase activator peptide mimicked it. Using mutant GluN2A overexpression, we show that Tyr-1292 and Tyr-1387 but not Tyr-1325 are responsible for the effect of leptin. Importantly, β-cells from db/db mice, a type 2 diabetes mouse model lacking functional leptin receptors, or from obese diabetic human donors failed to respond to leptin but hyperpolarized in response to NMDA. Our study reveals a signaling pathway wherein leptin modulates NMDARs via Src to regulate β-cell excitability and suggests NMDARs as a potential target to overcome leptin resistance.

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared with healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2 inhibitor treatment.