aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete, ß-arrestin-2-mediated SphK1-S1PR1-Akt signaling axis.

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via ß-arrestin-2 (ß-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a ß-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)-rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates ß-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on ß-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-ß-arr2-mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, ß-arr2-driven signaling pathways in caveolae.

Pulmonary transit time from contrast echocardiography and cardiac magnetic resonance imaging: Comparison between modalities and the impact of region of interest characteristics.

INTRODUCTION: The degree of correlation of pulmonary transit time (PTT) between contrast echocardiography and cardiac magnetic resonance imaging (MRI) across the spectrum of cardiac disease has not been quantified. In addition, the degree to which PTT estimates are affected by variation in location and size of regions of interest (ROI) is unknown. METHODS: Pulmonary transit time was obtained using an inflection point technique from individuals that underwent contrast echocardiography and cardiac MRI. Right ventricular, left atrial, and left ventricular ROIs were evaluated, and two sizes for each ROI were used. The Spearman correlation coefficient and Bland-Altman analysis were used for comparisons between modalities. Bland-Altman plots were also used to measure the impact of ROI size and location on transit times. RESULTS: Fourteen participants (age: 27-64 years; LV ejection fraction: 30%-60%) underwent both studies a median of 1 week apart. The correlation between modalities was significant for PTT (r = 0.65; P = 0.01) and normalized PTT (r = 0.80; P = 0.001). Cardiac MRI yielded transit times consistently higher than contrast echocardiography (bias ~ 1.4 seconds), but the discordance was not dependent on transit time magnitude. Low bias was observed for comparisons of ROI size and location (<0.5 seconds). CONCLUSIONS: Contrast echocardiography underestimates transit time measurements obtained by cardiac MRI, although the discrepancy was systematic and may have been contributed to by the interval between imaging studies. ROI location and size did not impact transit time values, suggesting that ROIs could be placed without intensive training, a step toward incorporation of real-time PTT measurement into echocardiographic laboratory workflow.

Myocardial T1 Measurement Predicts Beneficial LV Remodeling After Long-Term Heart Failure Therapy.

BACKGROUND: The myocardial longitudinal relaxation time (T1) on cardiac magnetic resonance imaging (CMR) can quantify myocardial fibrosis in the presence or absence of visually detectable late gadolinium (Gd) enhancement (LGE). Mineralocorticoid receptor antagonist (MRA) treatment produces beneficial remodeling in nonischemic dilated cardiomyopathy (NIDCM). We assessed the hypothesis that interstitial myocardial fibrosis measured with the use of CMR predicts left ventricular (LV) beneficial remodeling in NIDCM after heart failure (HF) treatment including MRAs. METHODS AND RESULTS: Twelve patients with NIDCM, on stable beta-blocker and angiotensin-converting enzyme inhibitor/angiotensin receptor-blocking therapy, were studied before and after 6-29 months of treatment with MRAs, by means of CMR assessment of LV structure, function, and T1 from standard Look-Locker sequences (T1LL). All patients had depressed cardiac function, dilated left ventricles, and no visual LGE. After adding MRA to HF treatment, the LV ejection fraction increased and the LV end-systolic volume index (LV end-systolic volume/m2) decreased in all patients (P < .0001). This this was inversely proportional to the baseline myocardial T1LL (r = -0.65; P = .02). CONCLUSION: Myocardial T1LL, in the absence of visually detectable LGE, was quantitatively related to the degree of beneficial LV remodeling achieved in response to adding MRA to a HF regimen.

An In Vitro Cynomolgus Vascular Surrogate System for Preclinical Drug Assessment and Human Translation.

OBJECTIVES: The predictive value of animal and in vitro systems for drug development is limited, particularly for nonhuman primate studies as it is difficult to deduce the drug mechanism of action. We describe the development of an in vitro cynomolgus macaque vascular system that reflects the in vivo biology of healthy, atheroprone, or advanced inflammatory cardiovascular disease conditions. APPROACH AND RESULTS: We compare the responses of the in vitro human and cynomolgus vascular systems to 4 statins. Although statins exert beneficial pleiotropic effects on the human vasculature, the mechanism of action is difficult to investigate at the tissue level. Using RNA sequencing, we quantified the response to statins and report that most statins significantly increased the expression of genes that promote vascular health while suppressing inflammatory cytokine gene expression. Applying computational pathway analytics, we identified statin-regulated biological themes, independent of cholesterol lowering, that provide mechanisms for off-target effects, including thrombosis, cell cycle regulation, glycogen metabolism, and ethanol degradation. CONCLUSIONS: The cynomolgus vascular system described herein mimics the baseline and inflammatory regional biology of the human vasculature, including statin responsiveness, and provides mechanistic insight not achievable in vivo.

Increased myocardial native T1 and extracellular volume in patients with Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) cardiomyopathy is a progressive disease for which there is no cure. Disease-specific therapies are needed that can be initiated before irreversible myocardial damage ensues. In order to evaluate therapeutic efficacy, surrogate endpoints other than ejection fraction must be found. The hypothesis of this study is that T1 and extracellular volume fraction (ECV) mapping using cardiovascular magnetic resonance (CMR) can detect diffuse extracellular matrix expansion in DMD patients with normal left ventricular ejection fraction (LVEF) and without myocardial late gadolinium enhancement (LGE). METHODS: Thirty-one DMD and 11 healthy control participants were prospectively enrolled. CMR using a modified Look-Locker (MOLLI) sequence was performed in all participants before and after contrast administration. T1 and ECV maps of the mid left ventricular myocardium were generated and regions of interest were contoured using the standard 6-segment AHA model. Global and segmental values were compared between DMD and controls using a Wilcoxon rank-sum test. RESULTS: The DMD participants had significantly higher mean native T1 compared with controls (1045 ms vs. 988 ms, p = 0.001). DMD participants with normal LVEF and without evidence of LGE also demonstrated elevated mean native T1 (1039 ms vs. 988 ms, p = 0.002, and 1038 ms vs. 988 ms, p = 0.011). DMD participants had a significantly greater mean ECV than controls (0.31 vs. 0.24, p < 0.001), even in the settings of normal LVEF (0.28 vs. 0.24, p < 0.001) and negative LGE (0.29 vs. 0.24, p = 0.001). CONCLUSIONS: DMD participants have elevated LV myocardial native T1 and ECV, even in the setting of normal LVEF and in the absence of LGE. T1 and ECV mapping in DMD have potential to serve as surrogate cardiomyopathy outcome measures for clinical trials.

Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than ß-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

Physiological responses related to increased grain yield under drought in the first biotechnology-derived drought-tolerant maize.

Maize (Zea mays ssp. mays L.) is highly susceptible to drought stress. This work focused on whole-plant physiological mechanisms by which a biotechnology-derived maize event expressing bacterial cold shock protein B (CspB), MON 87460, increased grain yield under drought. Plants of MON 87460 and a conventional control (hereafter 'control') were tested in the field under well-watered (WW) and water-limited (WL) treatments imposed during mid-vegetative to mid-reproductive stages during 2009-2011. Across years, average grain yield increased by 6% in MON 87460 compared with control under WL conditions. This was associated with higher soil water content at 0.5 m depth during the treatment phase, increased ear growth, decreased leaf area, leaf dry weight and sap flow rate during silking, increased kernel number and harvest index in MON 87460 than the control. No consistent differences were observed under WW conditions. This indicates that MON 87460 acclimated better under WL conditions than the control by lowering leaf growth which decreased water use during silking, thereby eliciting lower stress under WL conditions. These physiological responses in MON 87460 under WL conditions resulted in increased ear growth during silking, which subsequently increased the kernel number, harvest index and grain yield compared to the control.

Evaluation of post-contrast myocardial t1 in duchenne muscular dystrophy using cardiac magnetic resonance imaging.

The objective of the study was to perform a retrospective pilot study to evaluate the potential of myocardial T1 in assessment of Duchenne muscular dystrophy (DMD) cardiomyopathy. Early identification of DMD cardiac disease, particularly myocardial fibrosis, would allow earlier therapy, potentially improving outcomes. Shortened myocardial T1 measured by cardiac MRI (CMR) is a measure of cardiac fibrosis that may be detected before late gadolinium enhancement (LGE). We hypothesized that the post-contrast T1 obtained from the Look-Locker sequences (T1LL), an easily obtainable surrogate of myocardial T1, would be abnormally shortened in DMD compared with controls. T1LL measurement was performed on 21 DMD subjects and 11 controls; to account for individual variations in gadolinium distribution, myocardial T1LL was divided by blood pool T1LL, deriving T1LL ratios. DMD subjects had shorter mean T1LL ratio than controls (1.42 vs 1.72, p < 0.001). Subset analyses in DMD subjects with normal LVEF and without LGE also demonstrated significantly shorter T1LL ratio (-0.28, p < 0.001 and -0.25, p = 0.028). Post-contrast T1LL ratio is abnormally shortened in DMD compared with controls, even in DMD patients with otherwise normal CMRs. The application of more aggressive therapy for those with shorter T1LL may favorably alter morbidity and improve mortality associated with DMD cardiomyopathy. These data suggest that further prospective evaluation of myocardial T1 will be of benefit to patients with DMD.

An optimized fractionation method reveals insulin-induced membrane surface localization of GLUT1 to increase glycolysis in LßT2 cells.

Insulin is an important regulator of whole-body glucose homeostasis. In insulin sensitive tissues such as muscle and adipose, insulin induces the translocation of glucose transporter 4 (GLUT4) to the cell membrane, thereby increasing glucose uptake. However, insulin also signals in tissues that are not generally associated with glucose homeostasis. In the human reproductive endocrine axis, hyperinsulinemia suppresses the secretion of gonadotropins from gonadotrope cells of the anterior pituitary, thereby linking insulin dysregulation to suboptimal reproductive health. In the mouse, gonadotropes express the insulin receptor which has the canonical signaling response of IRS, AKT, and mTOR activation. However, the functional outcomes of insulin action on gonadotropes are unclear. Here, we demonstrate through use of an optimized cell fractionation protocol that insulin stimulation of the LßT2 gonadotropic cell line results in the unexpected translocation of GLUT1 to the plasma membrane. Using our high purity fractionation protocol, we further demonstrate that though Akt signaling in response to insulin is intact, insulin-induced translocation of GLUT1 occurs independently of Akt activation in LßT2 cells.

Impairment of subendocardial perfusion reserve and oxidative metabolism in nonischemic dilated cardiomyopathy.

BACKGROUND: Cardiac magnetic resonance (CMR) and [(11)C]acetate positron emission tomography (PET) were used to assess the hypothesis that patients with nonischemic dilated cardiomyopathy (NIDCM) have decreased subendocardial perfusion reserve and impaired oxidative metabolism, consistent with the concept of "energy starvation" in heart failure (HF). METHODS AND RESULTS: CMR myocardial perfusion was evaluated in 13 NIDCM patients and 15 control subjects with coronary risk factors and normal myocardial perfusion. The NIDCM patients underwent [(11)C]acetate PET. The myocardial perfusion index (MPI) was calculated as the normalized rate of myocardial signal augmentation following gadolinium contrast injection. Hyperemic transmural, subendocardial, and subepicardial MPI were reduced in NIDCM compared with control subjects [0.13 vs 0.18 (P < .001), 0.13 vs 0.17 (P < .001), and 0.13 vs 0.17 (P = .008), respectively]. The subendocardial perfusion reserve was 1.59 ± 0.21 vs 1.86 ± 0.32 for the subepicardium (P = .002), demonstrating reduced perfusion reserve. The myocardial oxidative metabolic rate (kmono) per unit demand (rate-pressure product) was reduced in proportion to perfusion reserve (P = .02) CONCLUSIONS: Impaired subendocardial perfusion reserve in NIDCM confirmed results previously attained only in animal models. Impaired perfusion and impaired oxidative metabolism are consistent with subendocardial energy starvation in HF.

The Culture and Justice Quorum at University of California, San Diego: A Departmental Approach to Dismantling Structural Racism.

PROBLEM: Structural racism is embedded within the structure and function of academic medical institutions. Although many institutions have begun to incorporate racial justice within academic medicine, it needs to be integral to every discipline and all aspects of medical education, research, and health system practice. Guidance is lacking, however, on how to create and sustain department-level action to shift culture and encourage antiracist work. APPROACH: To address the culture, uphold racial justice, and address the challenges of racism in medicine with dynamic and innovative solutions, the Department of Obstetrics, Gynecology and Reproductive Sciences at University of California, San Diego, formed a Culture and Justice Quorum (the Quorum) in September 2020. All department faculty, residents, fellows, and staff were invited to participate in the Quorum as ambassadors who commit to meet and facilitate Quorum work or as supporters who pledge Quorum support without regular meeting participation. OUTCOMES: In all, 153 of 155 invited individuals (98.7%) responded, with 36 (23.2%) requesting to participate as ambassadors and 117 (75.5%) as supporters. Quorum ambassadors have worked together to assess the climate of the department, university, and health system, including incorporating input and amplifying efforts of the department's resident leadership council. The Quorum has implemented initiatives to promote health equity and developed a report card to demonstrate activities, monitor progress, and ensure accountability. NEXT STEPS: Through the innovative Culture and Justice Quorum, the department aims to address structural racism, foster justice, and dismantle the foundational injustices embedded within departmental clinical, educational, and research work and within the wider culture. The Quorum offers a model for creating and sustaining department-level action to shift culture and encourage antiracist work. Since established, it has received institutional recognition, including receiving the 2022 Inclusive Excellent Award for Department-Organizational Unit, which recognizes outstanding institutional contributions for inclusion and diversity efforts.

Human cardiovascular disease model predicts xanthine oxidase inhibitor cardiovascular risk.

Some health concerns are often not identified until late into clinical development of drugs, which can place participants and patients at significant risk. For example, the United States Food and Drug Administration (FDA) labeled the xanthine oxidase inhibitor febuxostat with a"boxed" warning regarding an increased risk of cardiovascular death, and this safety risk was only identified during Phase 3b clinical trials after its approval. Thus, better preclinical assessment of drug efficacy and safety are needed to accurately evaluate candidate drug risk earlier in discovery and development. This study explored whether an in vitro vascular model incorporating human vascular cells and hemodynamics could be used to differentiate the potential cardiovascular risk associated with molecules that have similar on-target mechanisms of action. We compared the transcriptomic responses induced by febuxostat and other xanthine oxidase inhibitors to a database of 111 different compounds profiled in the human vascular model. Of the 111 compounds in the database, 107 are clinical-stage and 33 are FDA-labelled for increased cardiovascular risk. Febuxostat induces pathway-level regulation that has high similarity to the set of drugs FDA-labelled for increased cardiovascular risk. These results were replicated with a febuxostat analog, but not another structurally distinct xanthine oxidase inhibitor that does not confer cardiovascular risk. Together, these data suggest that the FDA warning for febuxostat stems from the chemical structure of the medication itself, rather than the target, xanthine oxidase. Importantly, these data indicate that cardiovascular risk can be evaluated in this in vitro human vascular model, which may facilitate understanding the drug candidate safety profile earlier in discovery and development.

Lipopolysaccharide-induced chronic inflammation increases female serum gonadotropins and shifts the pituitary transcriptomic landscape.

Introduction: Female reproductive function depends on a choreographed sequence of hormonal secretion and action, where specific stresses such as inflammation exert profound disruptions. Specifically, acute LPS-induced inflammation inhibits gonadotropin production and secretion from the pituitary, thereby impacting the downstream production of sex hormones. These outcomes have only been observed in acute inflammatory stress and little is known about the mechanisms by which chronic inflammation affects reproduction. In this study we seek to understand the chronic effects of LPS on pituitary function and consequent luteinizing and follicle stimulating hormone secretion. Methods: A chronic inflammatory state was induced in female mice by twice weekly injections with LPS over 6 weeks. Serum gonadotropins were measured and bulk RNAseq was performed on the pituitaries from these mice, along with basic measurements of reproductive biology. Results: Surprisingly, serum luteinizing and follicle stimulating hormone was not inhibited and instead we found it was increased with repeated LPS treatments. Discussion: Analysis of bulk RNA-sequencing of murine pituitary revealed paracrine activation of TGFß pathways as a potential mechanism regulating FSH secretion in response to chronic LPS. These results provide a framework with which to begin dissecting the impacts of chronic inflammation on reproductive physiology.

Global changes in gene expression during compatible and incompatible interactions of cowpea (Vigna unguiculata L.) with the root parasitic angiosperm Striga gesnerioides.

BACKGROUND: Cowpea, Vigna unguiculata L. Walp., is one of the most important food and forage legumes in the semi-arid tropics. While most domesticated forms of cowpea are susceptible to the root parasitic weed Striga gesnerioides, several cultivars have been identified that show race-specific resistance. Cowpea cultivar B301 contains the RSG3-301 gene for resistance to S. gesnerioides race SG3, but is susceptible to race SG4z. When challenged by SG3, roots of cultivar B301 develop a strong resistance response characterized by a hypersensitive reaction and cell death at the site of parasite attachment. In contrast, no visible response occurs in B301 roots parasitized by SG4z. RESULTS: Gene expression in the roots of the cowpea cultivar B301 during compatible (susceptible) and incompatible (resistant) interactions with S. gesnerioides races SG4z and SG3, respectively, were investigated at the early (6 days post-inoculation (dpi)) and late (13 dpi) stages of the resistance response using a Nimblegen custom design cowpea microarray. A total of 111 genes were differentially expressed in B301 roots at 6 dpi; this number increased to 2102 genes at 13 dpi. At 13 dpi, a total of 1944 genes were differentially expressed during compatible (susceptible) interactions of B301 with SG4z. Genes and pathways involved in signal transduction, programmed cell death and apoptosis, and defense response to biotic and abiotic stress were differentially expressed in the early resistance response; at the later time point, enrichment was primarily for defense-related gene expression, and genes encoding components of lignifications and secondary wall formation. In compatible interactions (B301-SG4z), multiple defense pathways were repressed, including those involved in lignin biosynthesis and secondary cell wall modifications, while cellular transport processes for nitrogen and sulfur were increased. CONCLUSION: Distinct changes in global gene expression profiles occur in host roots following successful and unsuccessful attempted parasitism by Striga. Induction of specific defense related genes and pathways defines components of a unique resistance mechanism. Some genes and pathways up-regulated in the host resistance response to SG3 are repressed in the susceptible interactions, suggesting that the parasite is targeting specific components of the host's defense. These results add to our understanding of plant-parasite interactions and the evolution of resistance to parasitic weeds.

Sexy gene conversions: locating gene conversions on the X-chromosome.

Gene conversion can have a profound impact on both the short- and long-term evolution of genes and genomes. Here, we examined the gene families that are located on the X-chromosomes of human (Homo sapiens), chimpanzee (Pan troglodytes), mouse (Mus musculus) and rat (Rattus norvegicus) for evidence of gene conversion. We identified seven gene families (WD repeat protein family, Ferritin Heavy Chain family, RAS-related Protein RAB-40 family, Diphosphoinositol polyphosphate phosphohydrolase family, Transcription Elongation Factor A family, LDOC1-related family, Zinc Finger Protein ZIC, and GLI family) that show evidence of gene conversion. Through phylogenetic analyses and synteny evidence, we show that gene conversion has played an important role in the evolution of these gene families and that gene conversion has occurred independently in both primates and rodents. Comparing the results with those of two gene conversion prediction programs (GENECONV and Partimatrix), we found that both GENECONV and Partimatrix have very high false negative rates (i.e. failed to predict gene conversions), which leads to many undetected gene conversions. The combination of phylogenetic analyses with physical synteny evidence exhibits high resolution in the detection of gene conversions.

Modification of ventriculo-arterial coupling by spironolactone in nonischemic dilated cardiomyopathy.

AIMS: We sought to clarify the role of ventriculo-arterial (V-A) coupling in the treatment of nonischemic dilated cardiomyopathy (NIDCM) by adding a mineralocorticoid receptor antagonist (MRA) to conventional anti-failure therapy. METHODS AND RESULTS: We employed cardiac magnetic resonance imaging to quantify left ventricular (LV) contractility and V-A coupling in normal subjects at rest (n = 11) and in patients with NIDCM (n = 12) before and after long term anti-failure therapy, in which MRA was added to conventional anti-failure therapy. After ≥6 months' treatment in NIDCM patients, LV volumes and mass decreased, and the LV ejection fraction increased from a median of 24% (17, 27) (interquartile range IQR) to 47 (42, 52) (P < 0.002), with a marked reduction in arterial elastance (Ea) from 2.89 mmHg/mL (2.34, 4.0) to 1.50 (1.29, 1.95) (P < 0.002), similar to Ea of normal subjects, 1.53 (1.34, 1.67) (P > 0.05). The V-A coupling ratio, Ea/end-systolic elastance (single-beat method), decreased by -1.08 (-1.96, -0.55), (P = 0.003), as did Ea/end-systolic pressure/end-systolic pressure ratio, -0.54 (0.35, 0.87), (P = 0.002). The preload recruitable stroke work (PRSW) increased as did PRSW indexed for Ea (both P = 0.002), which reflected 'total circulatory performance'. CONCLUSIONS: In NIDCM, adding MRA to conventional anti-failure therapy markedly improved LV ejection fraction and reduced peripheral vascular resistance, due to both improved LV contractility and especially to enhanced V-A coupling, as Ea decreased to normal. Total circulatory performance was a sensitive indicator of both LV pump performance and the arterial loading conditions.

Ketohexokinase inhibition improves NASH by reducing fructose-induced steatosis and fibrogenesis.

BACKGROUND & AIMS: Increasing evidence highlights dietary fructose as a major driver of non-alcoholic fatty liver disease (NAFLD) pathogenesis, the majority of which is cleared on first pass through the hepatic circulation by enzymatic phosphorylation to fructose-1-phosphate via the ketohexokinase (KHK) enzyme. Without a current approved therapy, disease management emphasises lifestyle interventions, but few patients adhere to such strategies. New targeted therapies are urgently required. METHODS: We have used a unique combination of human liver specimens, a murine dietary model of NAFLD and human multicellular co-culture systems to understand the hepatocellular consequences of fructose administration. We have also performed a detailed nuclear magnetic resonance-based metabolic tracing of the fate of isotopically labelled fructose upon administration to the human liver. RESULTS: Expression of KHK isoforms is found in multiple human hepatic cell types, although hepatocyte expression predominates. KHK knockout mice show a reduction in serum transaminase, reduced steatosis and altered fibrogenic response on an Amylin diet. Human co-cultures exposed to fructose exhibit steatosis and activation of lipogenic and fibrogenic gene expression, which were reduced by pharmacological inhibition of KHK activity. Analysis of human livers exposed to 13C-labelled fructose confirmed that steatosis, and associated effects, resulted from the accumulation of lipogenic precursors (such as glycerol) and enhanced glycolytic activity. All of these were dose-dependently reduced by administration of a KHK inhibitor. CONCLUSIONS: We have provided preclinical evidence using human livers to support the use of KHK inhibition to improve steatosis, fibrosis, and inflammation in the context of NAFLD. LAY SUMMARY: We have used a mouse model, human cells, and liver tissue to test how exposure to fructose can cause the liver to store excess fat and become damaged and scarred. We have then inhibited a key enzyme within the liver that is responsible for fructose metabolism. Our findings show that inhibition of fructose metabolism reduces liver injury and fibrosis in mouse and human livers and thus this may represent a potential route for treating patients with fatty liver disease in the future.

GNRH induces the unfolded protein response in the LbetaT2 pituitary gonadotrope cell line.

The neuropeptide GNRH 1 stimulates the secretion of the reproductive hormone LH in pituitary gonadotropes. Other secretory cell types depend on the unfolded protein response (UPR) pathway to regulate protein synthesis and protect against endoplasmic reticulum (ER) stress in response to differentiation or secretory stimuli. This study investigated the role of the UPR in GNRH action within the LbetaT2 gonadotrope model. Cells were treated with GNRH, and the activation of UPR signaling components and general translational status was examined. The ER-resident stress sensors, Atf6, Eif2ak3, and Ern1, are all present, and GNRH stimulation results in the phosphorylation of eukaryotic translation initiation factor 2A kinase 3 and its downstream effector, eukaryotic translation initiation factor 2A. Additionally, activation of the UPR was confirmed both in LbetaT2 as well as mouse primary pituitary cells through identifying GNRH-induced splicing of Xbp1 mRNA, a transcription factor activated by splicing by the ER stress sensor, ER to nucleus signaling 1. Ribosome profiling revealed that GNRH stimulation caused a transient attenuation in translation, a hallmark of the UPR, remodeling ribosomes from actively translating polysomes to translationally inefficient ribonucleoprotein complexes and monosomes. The transient attenuation of specific mRNAs was also observed. Overall, the results show that GNRH activates components of the UPR pathway, and this pathway may play an important physiological role in adapting the ER of gonadotropes to the burden of their secretory demand.

Androgen Suppresses In Vivo and In Vitro LH Pulse Secretion and Neural Kiss1 and Tac2 Gene Expression in Female Mice.

Androgens can affect the reproductive axis of both sexes. In healthy women, as in men, elevated exogenous androgens decrease gonad function and lower gonadotropin levels; such circumstances occur with anabolic steroid abuse or in transgender men (genetic XX individuals) taking androgen supplements. The neuroendocrine mechanisms by which endogenous or exogenous androgens regulate gonadotropin release, including aspects of pulsatile luteinizing hormone (LH) secretion, remain unknown. Because animal models are valuable for interrogating neural and pituitary mechanisms, we studied effects of androgens in the normal male physiological range on in vivo LH secretion parameters in female mice and in vitro LH secretion patterns from isolated female pituitaries. We also assessed androgen effects on hypothalamic and gonadotrope gene expression in female mice, which may contribute to altered LH secretion profiles. We used a nonaromatizable androgen, dihydrotestosterone (DHT), to isolate effects occurring specifically via androgen receptor (AR) signaling. Compared with control females, DHT-treated females exhibited markedly reduced in vivo LH pulsatility, with decreases in pulse frequency, amplitude, peak, and basal LH levels. Correlating with reduced LH pulsatility, DHT-treated females also exhibited suppressed arcuate nucleus Kiss1 and Tac2 expression. Separate from these neural effects, we determined in vitro that the female pituitary is directly inhibited by AR signaling, resulting in lower basal LH levels and reduced LH secretory responses to gonadotropin-releasing hormone pulses, along with lower gonadotropin gene expression. Thus, in normal adult females, male levels of androgen acting via AR can strongly inhibit the reproductive axis at both the neural and pituitary levels.

GLUT1-mediated glycolysis supports GnRH-induced secretion of luteinizing hormone from female gonadotropes.

The mechanisms mediating suppression of reproduction in response to decreased nutrient availability remain undefined, with studies suggesting regulation occurs within the hypothalamus, pituitary, or gonads. By manipulating glucose utilization and GLUT1 expression in a pituitary gonadotrope cell model and in primary gonadotropes, we show GLUT1-dependent stimulation of glycolysis, but not mitochondrial respiration, by the reproductive neuropeptide GnRH. GnRH stimulation increases gonadotrope GLUT1 expression and translocation to the extracellular membrane. Maximal secretion of the gonadotropin Luteinizing Hormone is supported by GLUT1 expression and activity, and GnRH-induced glycolysis is recapitulated in primary gonadotropes. GLUT1 expression increases in vivo during the GnRH-induced ovulatory LH surge and correlates with GnRHR. We conclude that the gonadotropes of the anterior pituitary sense glucose availability and integrate this status with input from the hypothalamus via GnRH receptor signaling to regulate reproductive hormone synthesis and secretion.