Faculty Bibliography

OBJECTIVES/OBJECTIVE:Brown adipose tissue (BAT) controls triglyceride-rich lipoprotein (TRL) catabolism. This process is mediated by the lipoprotein lipase (LPL), an enzyme that catalyzed the hydrolysis of triglyceride (TAG) in glycerol and fatty acids (FA), which are burned to generate heat. LPL activity is regulated by angiopoietin-like 4 (ANGPTL4), a secretory protein produced in adipose tissues (AT), liver, kidney, and muscle. While the role of ANGPTL4 in regulating lipoprotein metabolism is well established, the specific contribution of BAT derived ANGPTL4 in controlling lipid and glucose homeostasis is not well understood. METHODS AND RESULTS/RESULTS:We generated a novel mouse model lacking ANGPTL4 specifically in brown adipose tissue (BAT-KO). Here, we report that specific deletion of ANGPTL4 in BAT results in enhanced LPL activity, circulating TAG clearance and thermogenesis. Absence of ANGPTL4 in BAT increased FA oxidation and reduced FA synthesis. Importantly, we observed that absence of ANGPTL4 in BAT leads to a remarkable improvement in glucose tolerance in short-term HFD feeding. CONCLUSION/CONCLUSIONS:Our findings demonstrate an important role of BAT derived ANGPTL4 in regulating lipoprotein metabolism, whole-body lipid and glucose metabolism, and thermogenesis during acute cold exposure.

An equilibrium needs to be established by the cellular and acellular components of the ovarian follicle if developmental competence is to be acquired by the oocyte. Both cumulus cells (CCs) and follicular fluid (FF) are critical determinants for oocyte quality. Understanding how CCs and FF influence oocyte quality in the presence of deleterious systemic or pelvic conditions may impact clinical decisions in the course of managing infertility. Given that the functional integrities of FF and CCs are susceptible to concurrent pathological conditions, it is important to understand how pathophysiological factors influence natural fertility and the outcomes of pregnancy arising from the use of assisted reproduction technologies (ARTs). Accordingly, this review discusses the roles of CCs and FF in ensuring oocyte competence and present new insights on pathological conditions that may interfere with oocyte quality by altering the intrafollicular environment.

Objective: To determine whether monocyte-platelet aggregates (MPA) correlate with aortic aneurysm (AA) prevalence and severity. BACKGROUND: Inflammation and intraluminal thrombus are key hallmarks of complex AA. While monocytes fuel inflammation in AA, the contribution of platelets is unknown. We hypothesized that increased platelet activity yields to MPA that drive AA development and indicate disease severity.
Method(s): Blood was collected from 49 symptomatic patients admitted for aneurysm repair procedures (8 thoracic and 41 abdominal) and 36 matched controls. All subjects were on aspirin monotherapy. Platelet responsiveness to agonists was characterized by light transmission aggregometry. Flow cytometry analysis allowed leukocytes (CD45+)/monocytes (CD14+)-platelet (CD61+) aggregates (LPA/MPA) measurements in the blood and profiled MPA in post-surgical aneurysm tissues.
Result(s): Platelet aggregation in response to ADP (57% vs. 35% aggregation, p<0.001) and arachidonic acid (24% vs. 16% aggregation, p=0.03), was increased in patients with AA versus controls. LPA (17.7 vs 6.2% CD61+ leukocytes, p=0.002) and MPA (18.0 vs 7.2% CD61+ Monocytes, p=0.008) were robustly increased in AA vs controls. MPA but not LPA was strongly and positively associated with AA size (p<0.0001). To delve into the role of MPA in situ in AA sac, platelets and tissue macrophage activation was characterized. Compared to the non-diseased part of the aorta, diseased section had significantly higher platelet infiltration (7.0% vs 1.2% CD61+ cells, p=0.006) and interaction with CD68+ tissue macrophages (8.3% vs. 0.7%, CD61+ macrophages p=0.03). Notably, macrophages highly expressed the adhesion protein, ICAM-1, in the diseased part (39.6 vs 3.3% in the non-diseased section, p<0.001) which further increased to 69.4% (p=0.01) when macrophages were in contact with platelets.
Conclusion(s): Our data highlights MPA as a novel mediator valuable to predict AA prevalence and severity

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

Co-administration of sofosbuvir, an anti-hepatitis C virus medication, and antiarrhythmic amiodarone causes symptomatic severe bradycardia in patients and animal models. However, in a few in vitro studies, the combination of sofosbuvir and amiodarone resulted in tachycardiac effects in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). This discrepancy may be attributable to the use of immature hiPSC-CMs in the in vitro studies. To address this, we evaluated the ability of our in-house hiPSC-CMs to assess the interactions between sofosbuvir and amiodarone in vitro. We performed whole-cell patch recordings on hiPSC-CMs to examine the cardiac effect of sofosbuvir and amiodarone, alone or in combination. We found that sofosbuvir and amiodarone caused bradycardiac effects (the beating rate decreased to 75% of the vehicle control, P < 0.001) on our hiPSC-CMs when applied in combination, but they had no significant effect when applied alone. Furthermore, the bradycardiac effect was membrane potential dependent: it increased with depolarization. This raised the possibility that the bradycardiac effects in vivo may originate in nodal cells, which have a more depolarized resting membrane potential compared with ventricular cells. The bradycardiac effects of sofosbuvir plus amiodarone in vitro are consistent with the clinical phenotype and suggest that our hiPSC-CMs may serve as a useful tool in assessing cardiac safety during drug discovery and development process.

Background: KAT P channels couple cellular metabolism and electrophysiology. Their molecular composition varies in different tissues and species. Rodent atrial KAT P channels have the SUR1 regulatory subunit, are activated by diazoxide and have been implicated in arrhythmogenesis in hypertension and excess beta-adrenergic tone. In contrast, human atrial KATP channels are insensitive to diazoxide and modulate APD only during extreme metabolic stress, where the SUR2A regulatory subunit is thought to be predominant. Objective: We hypothesized that changes in the human atrial action potential associated with beta-agonism are mediated by recruitment of SUR1-containing KATP channels. Methods: We used human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes where expression of a fuorescent reporter is driven by the atrial-specifc gene sarcolipin. Atrial specifcation was induced with retinoic acid. Di-4-ANBDQBS was used to perform optical action potential measurements on days 65-80 of differentiation. Excised patch clamping was used to evaluate KAT P channel density. Heterozygous ABCC8 (SUR1+/-) cells were generated using CRISPR/CAS9. Results: Optical mapping data are for APD90 with stimulation at 1.25 Hz The combination of isoproterenol (ISO, 10mu M) and rolipram (ROL, 10mu M) abbreviated APD compared to control (247.4+/-12.5ms, n=16 vs 344.2+/-22.9ms, n=22; p=0.002). This was ameliorated by 10mu M glibenclamide (312.0+/-18.9ms, n=23 vs 247.4+/-12.5ms, n=16; p=0.01). More patches from cells exposed to ISO and ROL had functional KATP channels (4/22 vs 0/24, p=0.045). Diazoxide shortened APD (267.3+/-21.7ms, n=20 vs 344.2+/-22.9ms, n=22; p=0.02). This was potentiated by prior beta-agonism (179.7+/-14.3ms, n=18 vs 267.3+/-21.7ms, n=20; p=0.002). Deletion of one ABCC8 allele ameliorated APD shortening with exposure to ISO, ROL, and diazoxide (240.9+/-18.2ms, n=14 vs 179.7+/-14.3ms, n=18; p=0.012). Functional KATP channel density after exposure to beta-agonists was reduced in SUR1+/-cells (1/40 vs 4/22, p=0.049). Conclusion: SUR1-containing KATP channels partially mediate beta-adrenergic APD shortening in human atrial cells and may represent a therapeutic target for atrial arrhythmia prevention

p204, a murine member of the interferon-inducible p200 protein family, and its human analogue, IFI16, have been shown to function as tumor suppressors in vitro, but the molecular events involved, in particular in vivo, remain unclear. Herein we induced the Lewis Lung carcinoma (LLC) murine model of human lung cancer in p204 null mice (KO) and their control littermates (WT). We compared the transcriptome in spleen from WT and p204 KO mice using a high-throughput RNA-sequencing array. A total 30.02 Gb of clean data were obtained, and overall Q30% was greater than 90.54%. More than 75% of clean data from 12 transcriptome samples were mapped to exons. The results showed that only 11 genes exhibited altered expression in untreated p204 KO mice relative to untreated WT mice, while 393 altered genes were identified in tumor-bearing p204 KO mice when compared with tumor-bearing WT mice. Further differentially expressed gene cluster and gene ontology consortium classification revealed that numerous cytokines and their receptors, chemoattractant molecules, and adhesion molecules were significantly induced in p204 KO mice. This study provides novel insights to the p204 network in anti-tumor immune response and also presents a foundation for future work concerning p204-mediated gene expressions and pathways.

Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules, and are widely involved in the regulation of various physiological processes. The nematode C. elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans. However, the enzymes required for the last step in the production of many bioactive peptides - the carboxyterminal amidation reaction - have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in wild-type animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase (PHM) and a peptidylglycine α-amidating monooxygenase (PAM) had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans. All MS data is available via ProteomeXchange with identifier PXD008942. In summary, the key steps in neuropeptide-processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.

Tafazzin is the mitochondrial enzyme that catalyzes transacylation between a phospholipid and a lysophospholipid in remodeling. Mutations in tafazzin cause Barth syndrome, a potentially life-threatening disease with the major symptom being cardiomyopathy. In the tafazzin-deficient heart, cardiolipin (CL) acyl chains become abnormally heterogeneous unlike those in the normal heart with a single dominant linoleoyl species, tetralinoleoyl CL. In addition, the amount of CL decreases and monolysocardiolipin (MLCL) accumulates. Here we determine using high-resolution ³¹P nuclear magnetic resonance with cryoprobe technology the fundamental phospholipid composition, including the major but oxidation-labile plasmalogens, in the tafazzin-knockdown (TAZ-KD) mouse heart as a model of Barth syndrome. In addition to confirming a lower level of CL (6.4 ± 0.1 → 2.0 ± 0.4 mol % of the total phospholipid) and accumulation of MLCL (not detected → 3.3 ± 0.5 mol %) in the TAZ-KD, we found a substantial reduction in the level of plasmenylcholine (30.8 ± 2.8 → 18.1 ± 3.1 mol %), the most abundant phospholipid in the control wild type. A quantitative Western blot revealed that while the level of peroxisomes, where early steps of plasmalogen synthesis take place, was normal in the TAZ-KD model, expression of Far1 as a rate-determining enzyme in plasmalogen synthesis was dramatically upregulated by 8.3 (±1.6)-fold to accelerate the synthesis in response to the reduced level of plasmalogen. We confirmed lyso-plasmenylcholine or plasmenylcholine is a substrate of purified tafazzin for transacylation with CL or MLCL, respectively. Our results suggest that plasmenylcholine, abundant in linoleoyl species, is important in remodeling CL in the heart. Tafazzin deficiency thus has a major impact on the cardiac plasmenylcholine level and thereby its functions.