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Biomedical subjects

Megan R McMullen

Publications and source records attributed to Megan R McMullen.

10 recordsLinked to original sources
PubMed2024

Membrane-bound O-acyltransferase 7 (MBOAT7) shapes lysosomal lipid homeostasis and function to control alcohol-associated liver injury.

Recent genome-wide association studies (GWAS) have identified a link between single-nucleotide polymorphisms (SNPs) near the MBOAT7 gene and advanced liver diseases. Specifically, the common MBOAT7 variant (rs641738) associated with reduced MBOAT7 expression is implicated in non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis. However, the precise mechanism underlying MBOAT7-driven liver disease progression remains elusive. Previously, we identified MBOAT7-driven acylation of lysophosphatidylinositol lipids as key mechanism suppressing the progression of NAFLD (Gwag et al., 2019). Here, we show that MBOAT7 loss of function promotes ALD via reorganization of lysosomal lipid homeostasis. Circulating levels of MBOAT7 metabolic products are significantly reduced in heavy drinkers compared to healthy controls. Hepatocyte- (Mboat7-HSKO), but not myeloid-specific (Mboat7-MSKO), deletion of Mboat7 exacerbates ethanol-induced liver injury. Lipidomic profiling reveals a reorganization of the hepatic lipidome in Mboat7-HSKO mice, characterized by increased endosomal/lysosomal lipids. Ethanol-exposed Mboat7-HSKO mice exhibit dysregulated autophagic flux and lysosomal biogenesis, associated with impaired transcription factor EB-mediated lysosomal biogenesis and autophagosome accumulation. This study provides mechanistic insights into how MBOAT7 influences ALD progression through dysregulation of lysosomal biogenesis and autophagic flux, highlighting hepatocyte-specific MBOAT7 loss as a key driver of ethanol-induced liver injury.

Animals↗
PubMed2006

Chronic ethanol feeding increases activation of NADPH oxidase by lipopolysaccharide in rat Kupffer cells: role of increased reactive oxygen in LPS-stimulated ERK1/2 activation and TNF-alpha production.

Reactive oxygen species (ROS) contribute to the development of chronic ethanol-induced liver injury. Although ROS modulate the activity of many signal transduction pathways, the molecular targets of ROS during ethanol exposure are not well understood. Here, we investigated whether specific ROS-sensitive signal transduction pathways contribute to increased tumor necrosis factor alpha (TNF-alpha) production by Kupffer cells after chronic ethanol feeding to rats. Lipopolysaccharide (LPS) rapidly increased ROS production, measured by dihydrorhodamine fluorescence, in Kupffer cells from ethanol- and pair-fed rats, and ROS production was 2.5-fold greater in ethanol-fed compared with pair-fed. Pretreatment with diphenyleneiodonium (DPI), which inhibits reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, normalized ROS production in Kupffer cells from ethanol-fed rats. LPS rapidly increased Rac1-guanosinetriphosphatase (GTPase) activity and p67(phox) translocation to the plasma membrane in Kupffer cells from pair-fed rats. After ethanol feeding, Rac1-GTPase activity was already increased over pair-fed at baseline and remained elevated over pair-fed after LPS stimulation. Further, LPS-stimulated p67(phox) translocation to the plasma membrane was enhanced after chronic ethanol feeding. LPS-stimulated extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation, two signaling pathways regulated by ROS, were increased twofold in Kupffer cells from ethanol-fed rats compared with pair-fed controls. However, only LPS-stimulated ERK1/2 phosphorylation was inhibited by DPI, which also reduced LPS-stimulated TNF-alpha production in Kupffer cells from pair- and ethanol-fed rats. These results demonstrate that chronic ethanol feeding increases LPS-stimulated NADPH oxidase-dependent production of ROS in Kupffer cells. Further, ERK1/2 is an important target of NADPH oxidase-derived ROS in Kupffer cells, contributing to enhanced LPS-stimulated TNF-alpha production by Kupffer cells after chronic ethanol feeding.

Animals↗
PubMed2006

Adiponectin normalizes LPS-stimulated TNF-alpha production by rat Kupffer cells after chronic ethanol feeding.

Chronic ethanol feeding sensitizes Kupffer cells to activation by lipopolysaccharide (LPS), leading to increased production of tumor necrosis factor-alpha (TNF-alpha). Adiponectin treatment protects mice from ethanol-induced liver injury. Because adiponectin has anti-inflammatory effects on macrophages, we hypothesized that adiponectin would normalize chronic ethanol-induced sensitization of Kupffer cells to LPS-mediated signals. Serum adiponectin concentrations were decreased by 45% in rats fed an ethanol-containing diet for 4 wk compared with pair-fed rats. Adiponectin dose dependently inhibited LPS-stimulated accumulation of TNF-alpha mRNA and peptide in Kupffer cells from both pair- and ethanol-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to both globular (gAcrp) and full-length adiponectin (flAcrp) than Kupffer cells from pair-fed controls with suppression at 10 ng/ml adiponectin after chronic ethanol feeding. Kupffer cells expressed both adiponectin receptors 1 and 2; chronic ethanol feeding did not change the expression of adiponectin receptor mRNA or protein. gAcrp suppressed LPS-stimulated ERK1/2 and p38 phosphorylation as well as IkappaB degradation at 100-1,000 ng/ml in Kupffer cells from both pair- and ethanol-fed rats. However, only LPS-stimulated ERK1/2 phosphorylation was sensitive to 10 ng/ml gAcrp. gAcrp also normalized LPS-stimulated DNA binding activity of early growth response-1 with greater sensitivity in Kupffer cells from rats fed chronic ethanol. In conclusion, these results demonstrate that Kupffer cells from ethanol-fed rats are more sensitive to the anti-inflammatory effects of both gAcrp and flAcrp. Suppression of LPS-stimulated ERK1/2 signaling by low concentrations of gAcrp was associated with normalization of TNF-alpha production by Kupffer cells after chronic ethanol exposure.

Adiponectin↗
PubMed2006

Regulation of Kupffer cell activity during chronic ethanol exposure: role of adiponectin.

There is a growing appreciation that adipose tissue is a multifunctional organ. In addition to its central role in lipid storage, adipose tissue secretes a diverse group of proteins, called adipokines, involved in lipid metabolism, insulin sensitivity, angiogenesis etc. Adipocytes also secrete various inflammatory and anti-inflammatory mediators. Adiponectin, an adipokine with potent anti-inflammatory properties, is thought to play an important role in the regulation of inflammation. The development of alcoholic liver disease is thought to involve increased pro-inflammatory activity, mediated in part by the activation of Kupffer cells. Chronic ethanol feeding sensitizes Kupffer cells to activation by lipopolysaccharide (LPS), leading to increased production of reactive oxygen species (ROS) and tumor necrosis factor-alpha (TNF-alpha). Recent studies have demonstrated a hepato-protective effect of adiponectin in the progression of alcoholic liver disease. Herein are summarized recent data demonstrating that adiponectin treatment can normalize LPS-stimulated ROS production and TNF-alpha expression in Kupffer cells after chronic ethanol feeding. These studies suggest that the hepato-protective activity of adiponectin is due, at least in part, to a direct anti-inflammatory effect of adiponectin on Kupffer cells.

Adiponectin↗
PubMed2005

Early growth response-1 transcription factor is essential for ethanol-induced fatty liver injury in mice.

BACKGROUND & AIMS: Early growth response-1 (Egr-1), an immediate early gene/zinc-finger transcription factor, is required for maximal stimulation of tumor necrosis factor alpha (TNF-alpha) transcription in response to lipopolysaccharide (LPS). Because chronic ethanol exposure sensitizes macrophages to LPS-stimulated TNF-alpha expression, we have investigated the role of Egr-1 in mediating increased LPS-stimulated TNF-alpha expression after chronic ethanol feeding. Furthermore, because TNF-alpha contributes to alcoholic liver injury, we tested the hypothesis that Egr-1 is required for the development of ethanol-induced fatty liver injury in wild type and egr-1 -/- mice. METHODS: Wild-type and egr-1 -/- mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks. RESULTS: Wild-type mice fed the ethanol diet developed hepatic steatosis characterized by micro- and macrovesicular lipid accumulation. However, egr-1 -/- mice did not develop steatosis after ethanol feeding. Alanine transferase and TNF-alpha concentrations in serum were increased after ethanol feeding in wild-type but not egr-1 -/- mice. In wild-type mice, challenge with LPS increased Egr-1 messenger RNA (mRNA) and DNA binding activity in liver; this response to LPS was enhanced after chronic ethanol feeding. LPS challenge also increased hepatic TNF-alpha mRNA and serum TNF-alpha to a greater extent after ethanol feeding compared with pair-fed wild-type mice. However, chronic ethanol feeding did not enhance LPS-stimulated TNF-alpha mRNA or serum TNF-alpha in egr-1 -/- mice. CONCLUSIONS: These data show that Egr-1 contributes to increased LPS-mediated TNF-alpha expression after chronic ethanol and that the absence of Egr-1 prevents chronic ethanol-induced fatty liver, as well as increased sensitivity to LPS.

Animals↗
PubMed2003

Chronic ethanol exposure increases the binding of HuR to the TNFalpha 3'-untranslated region in macrophages.

Tumor necrosis factor alpha (TNFalpha) expression is a key mediator of ethanol-induced liver disease. Increased lipopolysaccharide (LPS)-stimulated TNFalpha expression in macrophages after chronic ethanol feeding is associated with a stabilization of TNFalpha mRNA (Kishore, R., McMullen, M. R., and Nagy, L. E. (2001) J. Biol. Chem. 276, 41930-41937). Here we show that the 3'-UTR of murine TNFalpha mRNA was sufficient to mediate increased LPS-stimulated expression of a luciferase reporter in RAW 264.7 macrophages after chronic ethanol exposure. Further, we show that HuR, a nuclear/cytoplasmic shuttling protein, which binds to TNFalpha mRNA, is required for increased expression of TNFalpha after chronic ethanol. In Kupffer cells, HuR was primarily localized to the nucleus and then translocated to the cytosol in response to LPS in both pair- and ethanol-fed rats. After chronic ethanol feeding, HuR quantity in the cytosol was greater, both at baseline and in response to LPS, compared with pair-fed controls. Using RNA gel shift assays, we found that LPS treatment increased HuR binding to the 65-nucleotide A + U-rich element of the TNFalpha 3'-UTR by 2-fold over baseline in Kupffer cells from pair-fed rats. After chronic ethanol feeding, HuR binding to the TNFalpha A + U-rich element was increased by more than 5-fold at baseline and in response to LPS, compared with pair-fed controls. Down-regulation of HuR expression by RNA interference prevented the chronic ethanol-induced increase in expression of luciferase reporters containing the TNFalpha 3'-UTR. Taken together, these data demonstrate that increased binding of HuR to the TNFalpha 3'-UTR contributes to increased LPS-stimulated TNFalpha expression in macrophages after chronic ethanol exposure.

3' Untranslated Regions↗
PubMed2002

Chronic ethanol increases lipopolysaccharide-stimulated Egr-1 expression in RAW 264.7 macrophages: contribution to enhanced tumor necrosis factor alpha production.

Increased production of tumor necrosis factor alpha (TNFalpha) is associated with the development of alcoholic liver disease. Culture of RAW264.7 macrophages with 25 mm ethanol for 48 h increased lipopolysaccharide (LPS)-stimulated accumulation of tumor necrosis factor alpha (TNFalpha) peptide and mRNA by 2-fold. We investigated whether chronic ethanol-induced increases in the DNA binding and/or promoter activity of the key transcription factors regulating LPS-stimulated TNFalpha promoter activity contribute to increased TNFalpha expression. Binding of Egr-1 to the TNFalpha promoter was increased by 2.5-fold after ethanol exposure, whereas NFkappaB binding was decreased to 30% of control. AP-1 binding was not affected. Changes in binding activity were paralleled by an increased contribution of the Egr-1 binding site and a decreased contribution of the NFkappaB site to LPS-stimulated TNFalpha promoter activity. Overexpression of dominant negative Egr-1 prevented the ethanol-induced increase in LPS-stimulated TNFalpha mRNA accumulation. Chronic ethanol exposure enhanced LPS-stimulated Egr-1 promoter-driven CAT expression and transcription of Egr-1. Induction of Egr-1 is dependent on ERK1/2 activation in other systems. Therefore, we investigated whether the ERK1/2 pathway mediated the chronic ethanol-induced increases in Egr-1 and TNFalpha. Increased Egr-1 promoter activity and TNFalpha mRNA accumulation after chronic ethanol were both prevented by overexpression of dominant negative ERK1/2. LPS-stimulated ERK1/2 phosphorylation was increased 2-fold in cells cultured with ethanol compared with controls. These results demonstrate that enhanced LPS-dependent activation of Egr-1 contributes to increased TNFalpha production after chronic ethanol exposure.

Animals↗
PubMed2002

Lipopolysaccharide stimulation of ERK1/2 increases TNF-alpha production via Egr-1.

Lipopolysaccharide (LPS) is a potent activator of tumor necrosis factor-alpha (TNF-alpha) production by macrophages. LPS stimulates the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and increases TNF-alpha mRNA and protein accumulation in RAW 264.7 murine macrophages. However, the role of ERK1/2 activation in mediating LPS-stimulated TNF-alpha production is not well understood. Inhibition of ERK1/2 activation with PD-98059 or overexpression of dominant negative ERK1/2 decreased LPS-induced TNF-alpha mRNA quantity. LPS rapidly increased early growth response factor (Egr)-1 binding to the TNF-alpha promoter; this response was blunted in cells treated with PD-98059 or transfected with dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase reporter gene linked to the Egr-1 promoter, we show that LPS increased Egr-1 promoter activity via an ERK1/2-dependent mechanism. These results delineate the role of ERK1/2 activation of Egr-1 activity in mediating LPS-induced increases in TNF-alpha mRNA expression in macrophages.

Animals↗
PubMed2002

ERK1/2 and Egr-1 contribute to increased TNF-alpha production in rat Kupffer cells after chronic ethanol feeding.

Activation of Kupffer cells by lipopolysaccharide (LPS) is a critical step in the pathogenesis of alcoholic liver disease. Kupffer cells isolated from rats fed ethanol in their diet for 4 wk accumulated 4.3-fold more tumor necrosis factor (TNF)-alpha in response to LPS compared with pair-fed rats. In contrast, LPS-stimulated interleukin (IL)-1 accumulation was 50% lower after ethanol feeding. LPS-stimulated TNF-alpha mRNA accumulation was twofold higher after ethanol feeding, whereas IL-1beta mRNA accumulation was blunted. To understand the mechanisms for this differential response, we investigated the effects of ethanol on LPS-dependent signal transduction. Chronic ethanol feeding increased LPS-stimulated extracellular receptor-activated kinases 1/2 (ERK1/2) activation. Activation of ERK1/2 was required for maximal increases in TNF-alpha and IL-1beta mRNA and was associated with increased binding of early growth response-1 (Egr-1) to the TNF-alpha promoter after ethanol feeding. In contrast, ethanol feeding completely abrogated activation of nuclear factor-kappaB DNA-binding activity by LPS and had no effect on AP-1 binding. Together, these data suggest that enhanced activation of ERK1/2 and Egr-1 contributes to increased TNF-alpha production after chronic ethanol feeding.

Alcoholism↗