Biology subjects

O'Dwyer, C.

Publications and source records attributed to O'Dwyer, C..

7 recordsLinked to original sources

bioRxiv2026

Casp1 and Ripk3 are required for homeostatic insulin secretion in mice

Objectives- Cell death and inflammatory pathways play important roles in adaptations to nutrient overload and metabolic dysfunction. This study investigates the metabolic consequences that arise from the dual disruption of both caspase 1 (Casp1) and receptor interacting protein kinase 3 (Ripk3) in mice fed a control or obesity-inducing diet. Methods- Male and female wild-type (WT), Casp1/11 knockout (KO), Ripk3 KO and Casp1/11/Ripk3 double knockout (DKO) mice were fed a matched low-fat or a 60% kcal high fat diet, followed by metabolic phenotyping. Islets were isolated from WT and DKO mice for measures of dynamic glucose-stimulated insulin and somatostatin (Sst) secretion. Islet architecture and cellular composition were assessed in WT and DKO mice by immunofluorescent staining of intact pancreatic sections. Pharmacological inhibition of Casp1 (Ac-YVAD-cmk) and Ripk3 (GSK872) was performed in WT and DKO mice using isolated islets and in vivo administration. Exogenous hormones were administered prior to glucose injection to test in vivo responses. Results- High-fat feeding resulted in increased adiposity in male, but not female mice, with single or double deletion of Casp1/11 and Ripk3. These mice also exhibited markers of impaired glucose tolerance and insulin sensitivity. Interestingly, when both Casp1 and Ripk3 were deleted or inhibited in mice fed a low-fat diet, mice experienced reductions in glucose excursion following administration of glucose due to increased plasma insulin levels. This increase in insulin secretion was recapitulated in isolated islets ex vivo and was independent of changes in the proportions of -, {beta}-, and {delta}-cells within the islet. There were significant reductions in the percentage of urocortin-3 (Ucn3)-positive {beta}-cells in DKO mice compared to control, suggesting altered Ucn3-Sst signaling; however, only exogenous Sst (Octreotide) and not Ucn3 was able to correct the decreased glucose excursion. Conclusions- Loss or inhibition of both Casp1 and Ripk3 fundamentally alter islet responses to glucose. Our findings highlight that endogenous Casp1 and Ripk3 act independently of inflammatory or cell death signals to coordinate normal glucose-stimulated insulin release.

physiology↗

bioRxiv2024

LAG3 marks activated but hyporesponsive NK cells.

Natural Killer (NK) cells are critical for immunosurveillance yet become dysfunctional in contexts such as chronic stimulation by viral infections or cancer. This phenomenon is similar to T cell exhaustion but less well characterized, which limits therapeutic interventions. As shown for T cells, NK cells often display an increased expression of immune checkpoint proteins (ICP) following chronic stimulation, and ICP blockade therapies are currently being explored for several cancer types, which have remarkable patient benefits. Nevertheless, the nature of ICP expression in NK cells is still poorly documented. In this study, we aimed to identify the conditions that lead to and the phenotype of immune checkpoint LAG3 (Lymphocyte-activation gene 3) expressing NK cells. Using various experimental models, we found that LAG3 is expressed by murine NK cells upon activation in different contexts, including in response to cancer and acute viral infections. LAG3 marks a subset of immature, proliferating and activated cells, which, despite activation, have a reduced capacity to respond to a broad range of stimuli. Further characterization also revealed that LAG3+ NK cells exhibit a transcriptional signature similar to that of exhausted CD8+ T cells. Taken together, our results support the use of LAG3 as a marker of dysfunctional NK cells across diverse chronic and acute inflammatory conditions.

immunology↗

bioRxiv2023

Thermoneutral housing does not accelerate metabolic dysfunction-associated fatty liver disease in male or female mice fed a Western diet

ObjectiveMetabolic dysfunction-associated fatty liver disease (MAFLD) represents a growing cause of mortality and morbidity and encompasses a spectrum of liver pathologies. Potential therapeutic targets have been identified and are currently being pre-clinically and clinically tested. However, while dozens of preclinical models have been developed to recapitulate various stages of MAFLD, few achieve fibrosis using an experimental design that mimics human pathogenesis. We sought to clarify whether the combination of thermoneutral (TN) housing and consumption of a classical Western diet (WD) would accelerate the onset of MAFLD and progression in male and female mice. MethodsMale and female C57Bl/6J mice were fed a nutrient-matched low-fat control or Western diet (41% Kcal from fat, 43% carbohydrate and 0.2% cholesterol; WD) starting at [~]12 wk of age for a further 16 wk. Mice were divided and housed with littermates at either standard temperature (TS; 22{degrees}C) or thermoneutral conditions (TN; [~]29{degrees}C). Mice underwent tests for glucose tolerance, insulin sensitivity and body composition, as well as intestinal permeability. Following tissue harvest, circulating and liver markers of hepatic disease progression toward steatosis and fibrosis were determined. ResultsWhile male mice housed at TN and fed a WD were significantly heavier than TS -housed control animals, no other differences in body weight or composition were observed. WD-fed females housed under TN conditions had higher circulating LDL-cholesterol; however, there were no other significant differences between TN and TS -housing in circulating or hepatic lipid levels. While WD-fed TN males had higher ALT levels, no other differences in markers of liver injury or disease progression were observed. Moreover, females housed at TN conditions and fed a WD remained significantly protected against the induction of fibrosis compared to male counterparts. Interestingly, sex-specific differences were observed in markers of glucose and insulin tolerance, where TN housing and WD feeding resulted in hyperglycemia and impaired insulin responsiveness in both sexes, but glucose intolerance only in male mice. ConclusionsWhile TN housing has been demonstrated to exacerbate high fat-induced hepatic steatosis and inflammation in male and female mice, coupling TN housing with a WD for 16 wk was not sufficient to augment fatty liver progression in male or female mice. HighlightsO_LIThermoneutral housing and Western diet feeding does not progress to NASH C_LIO_LIFemale mice are not more susceptible to obesity induced fatty liver under these conditions C_LIO_LITemperature and diet had sex-specific effects on glucose tolerance and insulin sensitivity C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/524609v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@9b9c5eorg.highwire.dtl.DTLVardef@36fc5aorg.highwire.dtl.DTLVardef@1f1b244org.highwire.dtl.DTLVardef@eea3df_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

Murine macrophage choline metabolism underpins IL-4 polarization and RELMα up-regulation

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated upregulation of choline transport compared to naive macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELM (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naive or mice infected with intestinal helminths Heligmosomoides polygyrus or Nippostrongylus brasiliensis were treated with the choline kinase inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELM expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus with increased egg burden, but there were no differences in intestinal worm count nor differences in N. brasiliensis parasite burden. Together, these data demonstrate that choline metabolism is required for macrophage RELM induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/510305v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@2f83a3org.highwire.dtl.DTLVardef@16b74b1org.highwire.dtl.DTLVardef@1ba1f8dorg.highwire.dtl.DTLVardef@6da06a_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

Myeloid deletion and therapeutic activation of AMP-activated protein kinase (AMPK) do not alter atherosclerosis in male or female mice

ObjectiveThe 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. Approach and ResultsWe aimed to clarify the role of myeloid-specific AMPK signaling by using LysM-Cre to drive the deletion of both the 1 and 2 catalytic subunits (MacKO), in male and female mice made acutely atherosclerotic by PCSK9-AAV and Western diet-feeding. After 6 weeks of Western diet feeding, half received daily injection of either the AMPK activator, A-769662 or a vehicle control for a further 6 weeks. After 12 weeks, myeloid cell populations were not different between genotype or sex. Similarly, aortic sinus plaque size, lipid staining and necrotic area were not different in male and female MacKO mice compared to their littermate floxed controls. Moreover, therapeutic intervention with A-769662 had no effect. There were no 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 or markers of autophagy showed no effect of either lacking AMPK signaling or systemic AMPK activation. ConclusionsOur data suggest that while defined roles for each catalytic AMPK subunit have been identified, global deletion of myeloid AMPK signaling does not significantly impact atherosclerosis. Moreover, we show that intervention with the first-generation AMPK activator, A-769662, was not able to stem the progression of atherosclerosis. Highlights- The deletion of both catalytic subunits of AMPK in myeloid cells has no significant effect on the progression of atherosclerosis in either male or female mice - Therapeutic delivery of a first-generation AMPK activator (A-769662) for the last 6 weeks of 12-week study had no beneficial effect in either male or female mice - Studying total AMPK deletion may mask specific effects of each isoform and highlights the need for targeted disruption of AMPK phosphorylation sites via knock-in mutations, rather than the traditional "sledgehammer" knockout approach

biochemistry↗

bioRxiv2019

Fatty acid-induced lipotoxicity inhibits choline metabolism independent of ER stress in mouse primary hepatocytes

Choline is an essential nutrient that is critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine and the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the CDP-choline or the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway. With the continuing global rise in the rates of obesity and non-alcoholic fatty liver disease, we sought to explore how excess fatty acids (FA), typical of an obesity and hepatic steatosis, affect choline uptake and metabolism in primary hepatocytes. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared to control. PE synthesis was slightly diminished; however, no compensatory changes in the PEMT pathway were observed. We next hypothesized that ER stress may be a potential mechanism by which palmitate treatment diminished choline. However, when we exposed primary hepatocytes to the common ER stress inducing compound tunicamycin, choline uptake, contrary to our expectation was augmented, concomitant with the transcript expression of choline transporters. Moreover, tunicamycin-induced ER stress divorced the observed increase in choline uptake from CDP-choline pathway flux since ER stress significantly diminished the incorporation and total PC content, similar to PE. Conclusion: Therefore, our results suggest that the altered FA milieu seen in obesity and fatty liver disease progression may adversely affect choline metabolism, but that compensatory mechanisms work to maintain phospholipid homeostasis.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC=\"FIGDIR/small/746750v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (42K):\norg.highwire.dtl.DTLVardef@1090f2aorg.highwire.dtl.DTLVardef@1c28eedorg.highwire.dtl.DTLVardef@35a80eorg.highwire.dtl.DTLVardef@ac5a53_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

In vitro hepatitis C virus infection and hepatic choline metabolism

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC) and can enter one carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses can impact phospholipid metabolism. In the current study we sought to interrogate the link between choline transport and early HCV infection. Namely, we aimed to investigate how HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing how the inhibition of choline uptake and metabolism upon concurrent HCV infection may alter early viral replication. Finally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter choline transporter-like family (CTL), and that CTL1 expression and the incorporation of choline into PC is diminished in 24 h infected FBS-cultured cells. Reciprocally, limiting the availability of choline for PC synthesis resulted in increased HCV replication at this early stage. In chronically HS-cultured Huh7.5 cells, there were no differences in the expression of choline transporters upon HCV infection or alterations to viral replication when choline transport was inhibited compared to control treatments. However, inhibiting choline uptake and metabolism in this system significantly impaired the production of infectious virions in HS-cultured cells. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC=\"FIGDIR/small/746776v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (56K):\norg.highwire.dtl.DTLVardef@133a652org.highwire.dtl.DTLVardef@1ec8fa8org.highwire.dtl.DTLVardef@1cb7626org.highwire.dtl.DTLVardef@b9bbc2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗