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.

Impaired Bile Secretion Promotes Hepatobiliary Injury in Sickle Cell Disease.

BACKGROUND AND AIMS: Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS: SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS: These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.

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.