An indispensable role for dynamin-related protein 1 in beige and brown adipogenesis.

Emerging evidence indicates that proper mitochondrial dynamics are critical for adipocyte differentiation and functional thermogenic capacity. We found that the mitochondrial fission protein dynamin-related protein 1 (DRP1, also known as DNML1) is highly expressed in brown adipose tissue compared to expression in white adipose tissue, and these expression levels increase during brown adipocyte differentiation. Our results reveal that the inhibition of DRP1 using mdivi-1 mitigates beige adipocyte differentiation and differentiation-associated mitochondrial biogenesis. We found that DRP1 is essential for the induction of the early-phase beige adipogenic transcriptional program. Intriguingly, inhibition of DRP1 is dispensable following the induction of beige adipogenesis and adipogenesis-associated mitochondrial biogenesis. Altogether, we demonstrate that DRP1 in preadipocytes plays an essential role in beige and brown adipogenesis.This article has an associated First Person interview with the first author of the paper.

Neutrophils Restrict Tumor-Associated Microbiota to Reduce Growth and Invasion of Colon Tumors in Mice.

BACKGROUND & AIMS: Neutrophils are among the most prevalent immune cells in the microenvironment of colon tumors; they are believed to promote growth of colon tumors, and their numbers correlate with outcomes of patients with colon cancer. Trials of inhibitors of neutrophil trafficking are underway in patients with cancer, but it is not clear how neutrophils contribute to colon tumorigenesis. METHODS: Colitis-associated colon cancer was induced in mice with conditional deletion of neutrophils (LysMCre;Mcl1fl/fl) and wild-type littermates (LysMCre;Mcl1wt/wt, control mice) by administration of azoxythmethane and/or dextran sulfate sodium. Sporadic colon tumorigenesis was assessed in neutrophil-deficient and neutrophil-replete mice with conditional deletion of colon epithelial Apc (Cdx2-CreERT2;Apcfl/fl). Primary colon tumor tissues from these mice were assessed by histology, RNA sequencing, quantitative polymerase chain reaction, and fluorescence in situ hybridization analyses. Fecal and tumor-associated microbiota were assessed by 16s ribosomal RNA sequencing. RESULTS: In mice with inflammation-induced and sporadic colon tumors, depletion of neutrophils increased the growth, proliferation, and invasiveness of the tumors. RNA sequencing analysis identified genes that regulate antimicrobial and inflammatory processes that were dysregulated in neutrophil-deficient colon tumors compared with colon tumors from control mice. Neutrophil depletion correlated with increased numbers of bacteria in tumors and proliferation of tumor cells, tumor-cell DNA damage, and an inflammatory response mediated by interleukin 17 (IL17). The 16s ribosomal RNA sequencing identified significant differences in the composition of the microbiota between colon tumors from neutrophil-deficient vs control mice. Administration of antibiotics or a neutralizing antibody against IL17 to neutrophil-deficient mice resulted in development of less-invasive tumors compared with mice given vehicle. We found bacteria in tumors to induce production of IL17, which promotes influx of intratumor B cells that promote tumor growth and progression. CONCLUSIONS: In comparisons of mice with vs without neutrophils, we found neutrophils to slow colon tumor growth and progression by restricting numbers of bacteria and tumor-associated inflammatory responses.

Role of Intestinal HIF-2α in Health and Disease.

The intestine is supported by a complex vascular system that undergoes dynamic and transient daily shifts in blood perfusion, depending on the metabolic state. Moreover, the intestinal villi have a steep oxygen gradient from the hypoxic epithelium adjacent to the anoxic lumen to the relative higher tissue oxygenation at the base of villi. Due to the daily changes in tissue oxygen levels in the intestine, the hypoxic transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α are essential in maintaining intestinal homeostasis. HIF-2α is essential in maintaining proper micronutrient balance, the inflammatory response, and the regenerative and proliferative capacity of the intestine following an acute injury. However, chronic activation of HIF-2α leads to enhanced proinflammatory response, intestinal injury, and colorectal cancer. In this review, we detail the major mechanisms by which HIF-2α contributes to health and disease of the intestine and the therapeutic implications of targeting HIF-2α in intestinal diseases.

Temporal induction of intestinal epithelial hypoxia-inducible factor-2α is sufficient to drive colitis.

Hypoxia is a notable feature of inflammatory bowel disease and chronic induction of hypoxia-inducible factor (HIF)-1α and HIF-2α (endothelial PAS domain protein 1, EPAS1) play important, but opposing, roles in its pathogenesis. While activation of HIF-1α decreases intestinal inflammation and is beneficial in colitis, activation of HIF-2α exacerbates colitis and increases colon carcinogenesis in animal models, primarily due to the role of epithelial HIF-2α in mounting a potent inflammatory response. Previous work from our laboratory showed that mice overexpressing intestinal epithelial HIF-2α led to massive intestinal inflammation and decreased survival. As oxygen homeostasis and HIFs are critical in embryonic development, it is not clear whether the observed intestinal inflammatory response was secondary to developmental defects. To address this question, the present study used a mouse model to temporally modulate expression of intestinal epithelial HIF-2α to assess its role in mediating inflammatory response. Remarkably, activation of HIF-2α in intestinal epithelial cells in adult mice increased expression of proinflammatory mediators; however, no decrease in survival was observed. Furthermore, in an acute model of colitis, activation of HIF-2α was sufficient to exacerbate colitis. These data confirm our previous finding that epithelial HIF-2α mediates inflammatory response and demonstrates that activation of HIF-2α is sufficient to exacerbate colitis.NEW & NOTEWORTHY Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disease of the intestinal tract. Hypoxia and activation of its downstream transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α are notable features of IBD. HIF-1α has well-characterized protective roles in IBD; however, the role of HIF-2α has been less studied. Using novel HIF-2α mouse models, we show that activation of HIF-2α in intestinal epithelial cells is sufficient to exacerbate colitis.

Natural Secretory Immunoglobulins Promote Enteric Viral Infections.

Noroviruses are enteric pathogens causing significant morbidity, mortality, and economic losses worldwide. Secretory immunoglobulins (sIg) are a first line of mucosal defense against enteric pathogens. They are secreted into the intestinal lumen via the polymeric immunoglobulin receptor (pIgR), where they bind to antigens. However, whether natural sIg protect against norovirus infection remains unknown. To determine if natural sIg alter murine norovirus (MNV) pathogenesis, we infected pIgR knockout (KO) mice, which lack sIg in mucosal secretions. Acute MNV infection was significantly reduced in pIgR KO mice compared to controls, despite increased MNV target cells in the Peyer's patch. Natural sIg did not alter MNV binding to the follicle-associated epithelium (FAE) or crossing of the FAE into the lymphoid follicle. Instead, naive pIgR KO mice had enhanced levels of the antiviral inflammatory molecules interferon gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) in the ileum compared to controls. Strikingly, depletion of the intestinal microbiota in pIgR KO and control mice resulted in comparable IFN-γ and iNOS levels, as well as MNV infectious titers. IFN-γ treatment of wild-type (WT) mice and neutralization of IFN-γ in pIgR KO mice modulated MNV titers, implicating the antiviral cytokine in the phenotype. Reduced gastrointestinal infection in pIgR KO mice was also observed with another enteric virus, reovirus. Collectively, our findings suggest that natural sIg are not protective during enteric virus infection, but rather, that sIg promote enteric viral infection through alterations in microbial immune responses.IMPORTANCE Enteric virus, such as norovirus, infections cause significant morbidity and mortality worldwide. However, direct antiviral infection prevention strategies are limited. Blocking host entry and initiation of infection provides an established avenue for intervention. Here, we investigated the role of the polymeric immunoglobulin receptor (pIgR)-secretory immunoglobulin (sIg) cycle during enteric virus infections. The innate immune functions of sIg (agglutination, immune exclusion, neutralization, and expulsion) were not required during control of acute murine norovirus (MNV) infection. Instead, lack of pIgR resulted in increased IFN-γ levels, which contributed to reduced MNV titers. Another enteric virus, reovirus, also showed decreased infection in pIgR KO mice. Collectively, our data point to a model in which sIg-mediated microbial sensing promotes norovirus and reovirus infection. These data provide the first evidence of the proviral role of natural sIg during enteric virus infections and provide another example of how intestinal bacterial communities indirectly influence MNV pathogenesis.

Maternal intestinal HIF-2α is necessary for sensing iron demands of lactation in mice.

The mechanisms that are essential for the maintenance of nutrient status in breast milk are unclear. Our data demonstrate that the intestine via hypoxia-inducible factor (HIF)-2α is an essential regulatory mechanism for maintaining the quality of breast milk. During lactation, intestinal HIF-2α is highly increased, leading to an adaptive induction of apical and basolateral iron transport genes. Disruption of intestinal HIF-2α (but not HIF-1α) or the downstream target gene divalent metal transporter (DMT)-1 in lactating mothers did not alter systemic iron homeostasis in the mothers, but led to anemia, decreased growth, and truncal alopecia in pups which was restored following weaning. Moreover, pups born from mothers with a disruption of intestinal HIF-2α led to long-term cognitive defects. Cross-fostering experiments and micronutrient profiling of breast milk demonstrated that the defects observed were due to decreased maternal iron delivery via milk. Increasing intestinal iron absorption by activation of HIF-2α or parenteral administration of iron-dextran in HIF-2α knockout mothers ameliorated anemia and restored neonatal development and adult cognitive functions. The present work details the importance of breast milk iron in neonatal development and uncovers an unexpected molecular mechanism for the regulation of nutritional status of breast milk through intestinal HIF-2α.

Fenofibrate Decreases Insulin Clearance and Insulin Secretion to Maintain Insulin Sensitivity.

High fat diet reduces the expression of CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a transmembrane glycoprotein that promotes insulin clearance and down-regulates fatty acid synthase activity in the liver upon its phosphorylation by the insulin receptor. Because peroxisome proliferator-activated receptor α (PPARα) transcriptionally suppresses CEACAM1 expression, we herein examined whether high fat down-regulates CEACAM1 expression in a PPARα-dependent mechanism. By activating PPARα, the lipid-lowering drug fenofibrate reverses dyslipidemia and improves insulin sensitivity in type 2 diabetes in part by promoting fatty acid oxidation. Despite reducing glucose-stimulated insulin secretion, fenofibrate treatment does not result in insulin insufficiency. To examine whether this is mediated by a parallel decrease in CEACAM1-dependent hepatic insulin clearance pathways, we fed wild-type and Pparα-/- null mice a high fat diet supplemented with either fenofibrate or Wy14643, a selective PPARα agonist, and examined their effect on insulin metabolism and action. We demonstrated that the decrease in insulin secretion by fenofibrate and Wy14643 is accompanied by reduction in insulin clearance in wild-type but not Pparα-/- mice, thereby maintaining normoinsulinemia and insulin sensitivity despite continuous high fat intake. Intact insulin secretion in L-CC1 mice with protected hepatic insulin clearance and CEACAM1 levels provides in vivo evidence that insulin secretion responds to changes in insulin clearance to maintain physiologic insulin and glucose homeostasis. These results also emphasize the relevant role of hepatic insulin extraction in regulating insulin sensitivity.

PPARα (Peroxisome Proliferator-activated Receptor α) Activation Reduces Hepatic CEACAM1 Protein Expression to Regulate Fatty Acid Oxidation during Fasting-refeeding Transition.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed at high levels in the hepatocyte, consistent with its role in promoting insulin clearance in liver. CEACAM1 also mediates a negative acute effect of insulin on fatty acid synthase activity. Western blot analysis reveals lower hepatic CEACAM1 expression during fasting. Treating of rat hepatoma FAO cells with Wy14,643, an agonist of peroxisome proliferator-activated receptor α (PPARα), rapidly reduces Ceacam1 mRNA and CEACAM1 protein levels within 1 and 2 h, respectively. Luciferase reporter assay shows a decrease in the promoter activity of both rat and mouse genes by Pparα activation, and 5'-deletion and block substitution analyses reveal that the Pparα response element between nucleotides -557 and -543 is required for regulation of the mouse promoter activity. Chromatin immunoprecipitation analysis demonstrates binding of liganded Pparα toCeacam1promoter in liver lysates ofPparα(+/+), but notPparα(-/-)mice fed a Wy14,643-supplemented chow diet. Consequently, Wy14,643 feeding reduces hepatic Ceacam1 mRNA and CEACAM1 protein levels, thus decreasing insulin clearance to compensate for compromised insulin secretion and maintain glucose homeostasis and insulin sensitivity in wild-type mice. Together, the data show that the low hepatic CEACAM1 expression at fasting is mediated by Pparα-dependent mechanisms. Changes in CEACAM1 expression contribute to the coordination of fatty acid oxidation and insulin action in the fasting-refeeding transition.

Increased Glucose-induced Secretion of Glucagon-like Peptide-1 in Mice Lacking the Carcinoembryonic Antigen-related Cell Adhesion Molecule 2 (CEACAM2).

Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with the Ceacam2 null mutation (Cc2(-/-)). This study investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in ß-cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-ß-cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2(-/-) islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2(-/-) mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared with wild type, Cc2(-/-) mice also showed a higher insulin excursion during the oral glucose tolerance test. Pretreating with exendin(9-39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag enteroendocrine cells was increased with siRNA-mediated Ceacam2 down-regulation in parallel to an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1-mediated mechanism, independent of confounding metabolic factors.

Intestine-specific Disruption of Hypoxia-inducible Factor (HIF)-2α Improves Anemia in Sickle Cell Disease.

Sickle cell disease (SCD) is caused by genetic defects in the ß-globin chain. SCD is a frequently inherited blood disorder, and sickle cell anemia is a common type of hemoglobinopathy. During anemia, the hypoxic response via the transcription factor hypoxia-inducible factor (HIF)-2α is highly activated in the intestine and is essential in iron absorption. Intestinal disruption of HIF-2α protects against tissue iron accumulation in iron overload anemias. However, the role of intestinal HIF-2α in regulating anemia in SCD is currently not known. Here we show that in mouse models of SCD, disruption of intestinal HIF-2α significantly decreased tissue iron accumulation. This was attributed to a decrease in intestinal iron absorptive genes, which were highly induced in a mouse model of SCD. Interestingly, disruption of intestinal HIF-2α led to a robust improvement in anemia with an increase in RBC, hemoglobin, and hematocrit. This was attributed to improvement in RBC survival, hemolysis, and insufficient erythropoiesis, which is evident from a significant decrease in serum bilirubin, reticulocyte counts, and serum erythropoietin following intestinal HIF-2α disruption. These data suggest that targeting intestinal HIF-2α has a significant therapeutic potential in SCD pathophysiology.

Intestinal HIF2α promotes tissue-iron accumulation in disorders of iron overload with anemia.

Several distinct congenital disorders can lead to tissue-iron overload with anemia. Repeated blood transfusions are one of the major causes of iron overload in several of these disorders, including ß-thalassemia major, which is characterized by a defective ß-globin gene. In this state, hyperabsorption of iron is also observed and can significantly contribute to iron overload. In ß-thalassemia intermedia, which does not require blood transfusion for survival, hyperabsorption of iron is the leading cause of iron overload. The mechanism of increased iron absorption in ß-thalassemia is unclear. We definitively demonstrate, using genetic mouse models, that intestinal hypoxia-inducible factor-2α (HIF2α) and divalent metal transporter-1 (DMT1) are activated early in the pathogenesis of ß-thalassemia and are essential for excess iron accumulation in mouse models of ß-thalassemia. Moreover, thalassemic mice with established iron overload had significant improvement in tissue-iron levels and anemia following disruption of intestinal HIF2α. In addition to repeated blood transfusions and increased iron absorption, chronic hemolysis is the major cause of tissue-iron accumulation in anemic iron-overload disorders caused by hemolytic anemia. Mechanistic studies in a hemolytic anemia mouse model demonstrated that loss of intestinal HIF2α/DMT1 signaling led to decreased tissue-iron accumulation in the liver without worsening the anemia. These data demonstrate that dysregulation of intestinal hypoxia and HIF2α signaling is critical for progressive iron overload in ß-thalassemia and may be a novel therapeutic target in several anemic iron-overload disorders.

Fatty acid binding protein-4 (FABP4) is a hypoxia inducible gene that sensitizes mice to liver ischemia/reperfusion injury.

BACKGROUND & AIMS: Fatty acid binding protein 4 (FABP4) has been known as a mediator of inflammatory response in the macrophages and adipose tissue, but its hepatic function is poorly understood. The goal of this study is to investigate the role of FABP4 in liver ischemia/reperfusion (I/R), a clinical condition that involves both hypoxia and inflammation. METHODS: To examine the I/R regulation of FABP4, mice were subjected to I/R surgery before being measured for FABP4 gene expression. Both loss-of-function (by using a pharmacological FABP4 inhibitor) and gain-of-function (by adenoviral overexpression of FABP4) were used to determine the functional relevance of FABP4 expression and its regulation during I/R. To determine the hypoxia responsive regulation of FABP4, primary mouse hepatocytes were exposed to hypoxia. The FABP4 gene promoter was cloned and its regulation by hypoxia inducible factor 1α (HIF-1α) was characterized by luciferase reporter gene, electrophoretic mobility shift, and chromatin immunoprecipitation assays. RESULTS: We found that the hepatic expression of FABP4 was markedly induced by I/R. At the functional level, pharmacological inhibition of FABP4 alleviated the I/R injury, whereas adenoviral overexpression of FABP4 sensitized mice to I/R injury. We also showed that exposure of primary hepatocytes to hypoxia or transgenic overexpression of HIF-1α in the mouse liver was sufficient to induce the expression of FABP4. Our promoter analysis established FABP4 as a novel transcriptional target of HIF-1α. CONCLUSIONS: FABP4 is a hypoxia inducible gene that sensitizes mice to liver I/R injury. FABP4 may represent a novel therapeutic target, and FABP4 inhibitors may be used as therapeutic agents to manage hepatic I/R injury.

Bacterial siderophores that evade or overwhelm lipocalin 2 induce hypoxia inducible factor 1α and proinflammatory cytokine secretion in cultured respiratory epithelial cells.

Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including enterobactin (Ent). However, Ent is bound by the host protein lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. Furthermore, the combination of Ent and Lcn2 (Ent+Lcn2) leads to enhanced secretion of interleukin-8 (IL-8) compared to that induced by either stimulus alone. Modified or structurally distinct siderophores, including yersiniabactin (Ybt) and glycosylated Ent (GlyEnt, or salmochelin), deliver iron to bacteria despite the presence of Lcn2. We hypothesized that the robust immune response to Ent and Lcn2 requires iron chelation rather than the Ent+Lcn2 complex itself and also can be stimulated by Lcn2-evasive siderophores. To test this hypothesis, cultured respiratory epithelial cells were stimulated with combinations of purified siderophores and Lcn2 and analyzed by gene expression microarrays, quantitative PCR, and cytokine immunoassays. Ent caused HIF-1α protein stabilization, induced the expression of genes regulated by hypoxia-inducible factor 1α (HIF-1α), and repressed genes involved in cell cycle and DNA replication, whereas Lcn2 induced expression of proinflammatory cytokines. Iron chelation by excess Ent or Ybt significantly increased Lcn2-induced secretion of IL-8, IL-6, and CCL20. Stabilization of HIF-1α was sufficient to enhance Lcn2-induced IL-6 secretion. These data indicate that respiratory epithelial cells can respond to bacterial siderophores that evade or overwhelm Lcn2 binding by increasing proinflammatory cytokine production.

Activation of HIF-1α does not increase intestinal tumorigenesis.

The hypoxic response is mediated by two transcription factors, hypoxia-inducible factor (HIF)-1α and HIF-2α. These highly homologous transcription factors are induced in hypoxic foci and regulate cell metabolism, angiogenesis, cell proliferation, and cell survival. HIF-1α and HIF-2α are activated early in cancer progression and are important in several aspects of tumor biology. HIF-1α and HIF-2α have overlapping and distinct functions. In the intestine, activation of HIF-2α increases inflammation and colon carcinogenesis in mouse models. Interestingly, in ischemic and inflammatory diseases of the intestine, activation of HIF-1α is beneficial and can reduce intestinal inflammation. HIF-1α is a critical transcription factor regulating epithelial barrier function following inflammation. The beneficial value of pharmacological agents that chronically activate HIF-1α is decreased due to the tumorigenic potential of HIFs. The present study tested the hypothesis that chronic activation of HIF-1α may enhance colon tumorigenesis. Two models of colon cancer were assessed, a sporadic and a colitis-associated colon cancer model. Activation of HIF-1α in intestinal epithelial cells does not increase carcinogenesis or progression of colon cancer. Together, the data provide proof of principle that pharmacological activation of HIF-1α could be a safe therapeutic strategy for inflammatory bowel disease.

Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis.

Background: Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms. Methods: We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays. Results: We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20. Conclusions: Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases.

Hepatocyte-specific EGFR deletion promotes fibrosis but has no effect on steatosis in fast food diet model of MASLD.

BACKGROUND & AIMS: MASLD has become the most prevalent chronic liver disorder, with no approved treatment. Our previous work demonstrated the efficacy of a pan-ErbB inhibitor, Canertinib, in reducing steatosis and fibrosis in a murine fast-food diet (FFD) model of MASLD. The current study explores the effects of hepatocyte-specific ErbB1 (i.e. EGFR) deletion in the FFD model. METHODS: EGFRflox/flox mice, treated with AAV8-TBG-CRE to delete EGFR specifically in hepatocytes (EGFR-KO), were fed either a chow-diet or FFD for 2 or 5-months. RESULTS: Hepatocyte-specific EGFR deletion reduced serum triglyceride levels but did not prevent steatosis. Surprisingly, hepatic fibrosis was increased in EGFR-KO mice in the long-term study, which correlated with activation of TGFß1/fibrosis signaling pathways. Further, nuclear levels of some of the major MASLD regulating transcription factors (SREBP1, PPARγ, PPARα, and HNF4α) were altered in FFD-fed EGFR-KO mice. Transcriptomic analysis revealed significant alteration of lipid metabolism pathways in EGFR-KO mice with changes in several relevant genes, including downregulation of fatty-acid synthase and induction of lipolysis gene, Pnpla2, without impacting overall steatosis. Interestingly, EGFR downstream signaling mediators, including AKT, remain activated in EGFR-KO mice, which correlated with increased activity pattern of other receptor tyrosine kinases, including ErbB3/MET, in transcriptomic analysis. Lastly, Canertinib treatment in EGFR-KO mice, which inhibits all ErbB receptors, successfully reduced steatosis, suggesting the compensatory roles of other ErbB receptors in supporting MASLD without EGFR. CONCLUSIONS: Hepatocyte-specific EGFR-KO did not impact steatosis, but enhanced fibrosis in the FFD model of MASLD. Gene-networks associated with lipid metabolism were greatly altered in EGFR-KO, but phenotypic effects might be compensated by alternate signaling-pathways.

Ceacam1 deletion causes vascular alterations in large vessels.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance and endothelial survival. However, its role in the morphology of macrovessels remains unknown. Mice lacking Ceacam1 (Cc1-/-) exhibit hyperinsulinemia, which causes insulin resistance and fatty liver. With increasing evidence of an association among hyperinsulinemia, fatty liver disease, and atherosclerosis, we investigated whether Cc1-/- exhibited vascular lesions in atherogenic-prone aortae. Histological analysis revealed impaired endothelial integrity with restricted fat deposition and aortic plaque-like lesions in Cc1-/- aortae, likely owing to their limited lipidemia. Immunohistochemical analysis indicated macrophage deposition, and in vitro studies showed increased leukocyte adhesion to aortic wall, mediated in part by elevation in vascular cell adhesion molecule 1 levels. Basal aortic eNOS protein and NO content were reduced, in parallel with reduced Akt/eNOS and Akt/Foxo1 phosphorylation. Ligand-induced vasorelaxation was compromised in aortic rings. Increased NADPH oxidase activity and plasma 8-isoprostane levels revealed oxidative stress and lipid peroxidation in Cc1-/- aortae. siRNA-mediated CEACAM1 knockdown in bovine aortic endothelial cells adversely affected insulin's stimulation of IRS-1/PI 3-kinase/Akt/eNOS activation by increasing IRS-1 binding to SHP2 phosphatase. This demonstrates that CEACAM1 regulates both endothelial cell autonomous and nonautonomous mechanisms involved in vascular morphology and NO production in aortae. Systemic factors such as hyperinsulinemia could contribute to the pathogenesis of these vascular abnormalities. Cc1-/- mice provide a first in vivo demonstration of distinct CEACAM1-dependent hepatic insulin clearance linking hepatic to macrovascular abnormalities.

Oxidative Stress and Redox Signaling in the Pathophysiology of Liver Diseases.

The increased production of derivatives of molecular oxygen and nitrogen in the form of reactive oxygen species (ROS) and reactive nitrogen species (RNS) lead to molecular damage called oxidative stress. Under normal physiological conditions, the ROS generation is tightly regulated in different cells and cellular compartments. Any disturbance in the balance between the cellular generation of ROS and antioxidant balance leads to oxidative stress. In this article, we discuss the sources of ROS (endogenous and exogenous) and antioxidant mechanisms. We also focus on the pathophysiological significance of oxidative stress in various cell types of the liver. Oxidative stress is implicated in the development and progression of various liver diseases. We narrate the master regulators of ROS-mediated signaling and their contribution to liver diseases. Nonalcoholic fatty liver diseases (NAFLD) are influenced by a "multiple parallel-hit model" in which oxidative stress plays a central role. We highlight the recent findings on the role of oxidative stress in the spectrum of NAFLD, including fibrosis and liver cancer. Finally, we provide a brief overview of oxidative stress biomarkers and their therapeutic applications in various liver-related disorders. Overall, the article sheds light on the significance of oxidative stress in the pathophysiology of the liver. © 2022 American Physiological Society. Compr Physiol 12:3167-3192, 2022.

Emerging Role of Hepatic Ketogenesis in Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD), the most common chronic liver diseases, arise from non-alcoholic fatty liver (NAFL) characterized by excessive fat accumulation as triglycerides. Although NAFL is benign, it could progress to non-alcoholic steatohepatitis (NASH) manifested with inflammation, hepatocyte damage and fibrosis. A subset of NASH patients develops end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. The pathogenesis of NAFLD is highly complex and strongly associated with perturbations in lipid and glucose metabolism. Lipid disposal pathways, in particular, impairment in condensation of acetyl-CoA derived from ß-oxidation into ketogenic pathway strongly influence the hepatic lipid loads and glucose metabolism. Current evidence suggests that ketogenesis dispose up to two-thirds of the lipids entering the liver, and its dysregulation significantly contribute to the NAFLD pathogenesis. Moreover, ketone body administration in mice and humans shows a significant improvement in NAFLD. This review focuses on hepatic ketogenesis and its role in NAFLD pathogenesis. We review the possible mechanisms through which impaired hepatic ketogenesis may promote NAFLD progression. Finally, the review sheds light on the therapeutic implications of a ketogenic diet in NAFLD.

Vertical sleeve gastrectomy increases duodenal Lactobacillus spp. richness associated with the activation of intestinal HIF2α signaling and metabolic benefits.

OBJECTIVE: Vertical Sleeve Gastrectomy (VSG) is one of the most efficacious treatments for obesity and its comorbidities. Although a range of evidence suggests that alterations of the microbiota in the distal gut following VSG are pivotal to these metabolic improvements, the effect of surgery to alter the microbiota of the proximal intestine and its effect on host physiology remain largely unknown. As the main bacteria in the upper small intestine, Lactobacillus subspecies have been appreciated as important regulators of gut function. These bacteria also regulate intestinal Hypoxia- Inducible Factor 2α (HIF2α) signaling that plays an integral role in gut physiology and iron absorption. In the present study, we sought to determine the impact of VSG on Lactobacillus spp. in the small intestine and potential downstream impacts of Lactobacillus spp. on HIF2α, specifically in the duodenum. METHODS: To determine the effects of VSG on the microbiota and HIF2α signaling in the duodenum, VSG surgeries were performed on diet-induced obese mice. To further probe the relationship between Lactobacillus spp. and HIF2α signaling in the duodenum, we applied a customized high-fat but iron-deficient diet on mice to increase duodenal HIF2α signaling and determined alterations of gut bacteria. To explore the causal role of Lactobacillus spp. in duodenal HIF2α signaling activation, we chronically administered probiotics containing Lactobacillus spp. to high-fat-fed obese mice. Lastly, we studied the effect of lactate, the major metabolite of Lactobacilli, on HIF2α in ex vivo duodenal organoids. RESULTS: There were pronounced increases in the abundance of Lactobacillus spp. in samples isolated from duodenal epithelium in VSG-operated mice as compared to sham-operated mice. This was accompanied by an increase in the expression of genes that are targets of HIF2α in the duodenum of VSG-treated mice. Activating HIF2α signaling with a high-fat but iron-deficient diet resulted in weight loss, improvements in glucose regulation, and increased Lactobacillus spp. richness in the duodenum as compared to mice on an iron-replete diet. Chronic administration of probiotics containing Lactobacillus spp. not only increased HIF2α signaling in the duodenum such as occurs after VSG but also resulted in reduced weight gain and improved glucose tolerance in high-fat-fed mice. Furthermore, lactate was able to activate HIF2α in ex vivo duodenal organoids. CONCLUSIONS: These results support a model whereby VSG increases duodenal Lactobacillus richness and potentially stimulates intestinal HIF2α signaling via increased lactate production.