Tissue-specific Knockout Mice Research Articles

Adipose Triglyceride Lipase–Mediated Adipocyte Lipolysis Exacerbates Acute Pancreatitis Severity in Mouse Models and Patients

Dyslipolysis of adipocytes has played a critical role in various diseases. Adipose triglyceride lipase (ATGL) is a rate-limiting enzyme in adipocyte autonomous lipolysis. However, whether the degree of adipocyte lipolysis relates to the prognoses in acute pancreatitis (AP) and the role of ATGL mediated lipolysis in the pathogenesis of AP remains elusive. The visceral adipose tissue (VAT) consumption rate (VATR) in the acute stage was measured in both AP patients and mouse models. Lipolysis levels and ATGL expression were detected in caerulein (CER)-induced AP models. CL316,243, a lipolysis stimulator, and adipose tissue-specific ATGL knockout (AAKO) mice were used to further investigate the role of lipolysis in AP. The ATGL-specific inhibitor, Atglistatin, was performed in C57Bl/6N and ob/ob AP models. This study found that increased VATR in the acute phase was independently associated with adverse prognoses in AP patients, which was validated in mice AP models. Lipolysis of adipocytes was elevated in AP mice. Stimulation of lipolysis could aggravate AP. Genetic blockage of ATGL specifically in adipocytes was able to alleviate the damage to AP. The application of Atglistatin could effectively protect against AP in both lean and obese mice. These findings demonstrated that ATGL-mediated adipocyte lipolysis exacerbates AP and highlighted the therapeutic potential of ATGL as a drug target for AP.

Controlling autoimmune diabetes onset by targeting Protease-Activated Receptor 2

BackgroundType 1 diabetes (T1D) is a challenging autoimmune disease, characterized by an immune system assault on insulin-producing β-cells. As insulin facilitates glucose absorption into cells and tissues, β-cell deficiency leads to elevated blood glucose levels on one hand and target-tissues starvation on the other. Despite efforts to halt β-cell destruction and stimulate recovery, success has been limited. Our recent investigations identified Protease-Activated Receptor 2 (Par2) as a promising target in the battle against autoimmunity. We discovered that Par2 activation’s effects depend on its initial activation site: exacerbating the disease within the immune system but fostering regeneration in affected tissue. MethodsWe utilized tissue-specific Par2 knockout mice strains with targeted Par2 mutations in β-cells, lymphocytes, and the eye retina (as a control) in the NOD autoimmune diabetes model, examining T1D onset and β-cell survival. ResultsWe discovered that Par2 expression within the immune system accelerates autoimmune processes, while its presence in β-cells offers protection against β-cell destruction and T1D onset. This suggests a dual-strategy treatment for T1D: inhibiting Par2 in the immune system while activating it in β-cells, offering a promising strategy for T1D. ConclusionsThis study highlights Par2's potential as a drug target for autoimmune diseases, particularly T1D. Our results pave the way for precision medicine approaches in treating autoimmune conditions through targeted Par2 modulation.

Abstract 3016: MCPIP1 endoribonuclease protects from liver fibrosis and regulates hepatocellular development

Abstract Introduction: MCPIP1 (Monocyte Chemotactic Protein-1 Induced Protein), also known as Regnase-1, belongs to the CCCH zinc finger protein family, featuring a PilT N-terminus (PIN) domain-like RNase domain. Utilizing its intrinsic endonuclease activity, MCPIP1 functions as a precise modulator of cytokines, exhibiting an anti-inflammatory effect through the targeted degradation of specific mRNA transcripts. While recent studies have highlighted MCPIP1 as a potential tumor suppressor with decreased levels in breast cancer and clear cell renal cell carcinoma, its role in hepatocellular carcinoma (HCC) development remains unexplored. Material and Methods: To investigate the role of MCPIP1 in HCC development, we used tissue-specific knockout mice with the deletion of Mcpip1 in hepatocytes. HCC was induced by administering diethylnitrosamine (DEN) to two-week-old mice. We conducted a comprehensive analysis, including the assessment of EMT and fibrosis markers, proinflammatory cytokines, and immune cell infiltration, using Western blot (WB), mass spectrometry, quantitative PCR (qPCR), and multiple immunohistochemistry (IHC) stainings. To identify differences between the wild-type (wt) and Mcpip1 knockout (KO) mice groups more effectively, hepatocytes were isolated using the perfusion method, and RNA-Seq analysis was performed. Results and Discussion: The tissue-specific knockout of Mcpip1 in the liver has notable molecular consequences. Transcriptomic profiling revealed an upregulation of transcripts integral to tumor development, including Cxcr4, Met, Cxcl12, Hgf, and Src, alongside factors characteristically associated with cancer stem cells, Myc, Klf4, and CD133. The absence of MCPIP1 triggered immune cell activation in the liver, as evidenced by an augmented expression of markers specific to myeloid and lymphoid cell populations, indicative of an intensified immunological response. IHC examination of liver tissue from Mcpip1-deficient mice revealed significant fibrotic alterations, characterized by increased collagen deposition, and enhanced expression of alpha-smooth muscle actin (αSMA). The lack of Mcpip1 influences the increased levels of vimentin, fibronectin, and connective tissue growth factor (cTGF), indicating enhanced tissue remodeling. Conclusions: MCPIP1, recognized for its anti-inflammatory functions, appears to function as a potential tumor suppressor in HCC development. The lack of MCPIP1 results in the upregulation of transcripts linked to tumorigenesis, immune cell activation, and notable fibrotic changes, highlighting its involvement in the complex molecular dynamics of HCC progression. This study was supported by National Science Center grants no. 2017/26/E/NZ5/00691, 2021/41/N/NZ4/04187. Citation Format: Katarzyna Miekus, Oliwia Kwapisz, Paulina Marona, Judyta Gorka, Jerzy Kotlinowski, Ewelina Pospiech, Jolanta Jura. MCPIP1 endoribonuclease protects from liver fibrosis and regulates hepatocellular development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3016.

Immune cell-intrinsic Ah receptor facilitates the expression of antimicrobial REG3G in the small intestine.

The intestinal epithelial layer is susceptible to damage by chemical, physiological and mechanical stress. While it is essential to maintain the integrity of epithelium, the biochemical pathways that contribute to the barrier function have not been completely investigated. Here we demonstrate an aryl hydrocarbon receptor (AHR)-dependent mechanism facilitating the production of the antimicrobial peptide AMP regenerating islet-derived protein 3 gamma (REG3G), which is essential for intestinal homeostasis. Genetic ablation of AHR in mice impairs pSTAT3-mediated REG3G expression and increases bacterial numbers of Segmented filamentous bacteria (SFB) and Akkermansia muciniphila in the small intestine. Studies with tissue-specific conditional knockout mice revealed that the presence of AHR in the epithelial cells of the small intestine is not required for the production of REG3G through the phosphorylated STAT3-mediated pathway. However, immune-cell-specific AHR activity is necessary for normal expression of REG3G in all regions of the small intestine. A diet rich in broccoli, capable of inducing AHR activity, increases REG3G production when compared to a semi-purified diet that is devoid of ligands that can potentially activate the AHR, thus highlighting the importance of AHR in antimicrobial function. Overall, these data suggest that homeostatic antimicrobial REG3G production is increased by an AHR pathway intrinsic to the immune cells in the small intestine.

Abstract B012: Wnt/Ror2 signaling regulates myoepithelial cell fate and luminal cell proliferation during mammary development

Abstract Background: The mammary gland exhibits extensive proliferative and differentiation potential throughout development. Wnt signaling has been recognized as a crucial player in multiple stages of mammary gland development, including mammary placode specification, gland branching morphogenesis, stem cell maintenance, and alveolar development. In our laboratory, prior research has revealed the importance of the β-catenin-independent noncanonical Wnt receptor, Ror2, in branching morphogenesis, cell differentiation, and actin-cytoskeletal dynamics within the mammary epithelium. However, the specific roles of Ror2 in the myoepithelial and luminal cell compartments have yet to be fully elucidated. Methods and Results: In this study, we investigated the functional role of Ror2 in these two distinct cell populations by developing mouse models with lineage-specific deletion of Ror2 in myoepithelial versus luminal epithelial compartments. To achieve this, we generated two tamoxifen-inducible, p63- and Keratin 8-specific Ror2 knockout mouse models, which carried a ROSA26-mT/mG fluorescent reporter for mammary myoepithelial cells and luminal cells, respectively. We induced postnatal Ror2 deletion at week 4 or 6 and assessed the mammary glands 2 to 4 weeks after Ror2 deletion. Through whole-mount imaging of mammary glands, we observed a significant increase in branching morphogenesis when Ror2 was deleted in the myoepithelial compartment, which was further confirmed in 3D branching assays in vitro. RNA-sequencing analysis revealed novel transcriptional alterations encompassing altered morphogenesis, cell differentiation, and intermediate filament organization upon Ror2 loss. Additional gene expression validation and immunofluorescence demonstrated altered myoepithelial cell fate specification, leading to a shift to a luminal-like cell fate, along with altered cell-cell adhesion among epithelial populations and abnormal branching morphogenesis within the developing gland. Moreover, luminal-specific deletion of Ror2 resulted in increased proliferation and abnormal lumen formation, both in vivo and in vitro. Conclusion: Our findings from these tissue-specific knockout mouse models highlight the critical role of the noncanonical Wnt receptor, Ror2, in regulating mammary epithelial cell fate, branching morphogenesis, and proliferation. These results also provide intriguing insights into the coordination between canonical and noncanonical Wnt signaling in mammary gland development, with potential implications for understanding breast cancer tumorigenesis and heterogeneity (Supported by NIH CA016303-46 and CPRIT RP210027). Citation Format: Hongjiang Si, Jeffrey Rosen, Chad Creighton, Kevin Roarty. Wnt/Ror2 signaling regulates myoepithelial cell fate and luminal cell proliferation during mammary development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B012.

Zinc finger protein ZNF638 regulates triglyceride metabolism via ANGPTL8 in an estrogen dependent manner

Background and aimTriglyceride (TG) levels are closely related to obesity, fatty liver and cardiovascular diseases, while the regulatory factors and mechanism for triglyceride homeostasis are still largely unknown. Zinc Finger Protein 638 (ZNF638) is a newly discovered member of zinc finger protein family for adipocyte function in vitro. The aim of the present work was to investigate the role of ZNF638 in regulating triglyceride metabolism in mice. MethodsWe generated ZNF638 adipose tissue specific knockout mice (ZNF638 FKO) by cross-breeding ZNF638 flox to Adiponectin-Cre mice and achieved adipose tissue ZNF638 overexpression via adenoviral mediated ZNF638 delivery in inguinal adipose tissue (iWAT) to examined the role and mechanisms of ZNF638 in fat biology and whole-body TG homeostasis. ResultsAlthough ZNF638 FKO mice showed similar body weights, body composition, glucose metabolism and serum parameters compared to wild-type mice under chow diet, serum TG levels in ZNF638 FKO mice were increased dramatically after refeeding compared to wild-type mice, accompanied with decreased endothelial lipoprotein lipase (LPL) activity and increased lipid absorption of the small intestine. Conversely, ZNF638 overexpression in iWAT reduced serum TG levels while enhanced LPL activity after refeeding in female C57BL/6J mice and obese ob/ob mice. Specifically, only female mice exhibited altered TG metabolism upon ZNF638 expression changes in fat. Mechanistically, RNA-sequencing analysis revealed that the TG regulator angiopoietin-like protein 8 (Angptl8) was highly expressed in iWAT of female ZNF638 FKO mice. Neutralizing circulating ANGPTL8 in female ZNF638 FKO mice abolished refeeding-induced TG elevation. Furthermore, we demonstrated that ZNF638 functions as a transcriptional repressor by recruiting HDAC1 for histone deacetylation and broad lipid metabolic gene suppression, including Angptl8 transcription inhibition. Moreover, we showed that the sexual dimorphism is possibly due to estrogen dependent regulation on ZNF638-ANGPTL8 axis. ConclusionWe revealed a role of ZNF638 in the regulation of triglyceride metabolism by affecting Angptl8 transcriptional level in adipose tissue with sexual dimorphism.

Requirement of Site-Specific Tyrosine Phosphorylation of Cortactin in Retinal Neovascularization and Vascular Leakage.

Retinal neovascularization is a major cause of vision impairment. Therefore, the purpose of this study is to investigate the mechanisms by which hypoxia triggers the development of abnormal and leaky blood vessels. A variety of cellular and molecular approaches as well as tissue-specific knockout mice were used to investigate the role of Cttn (cortactin) in retinal neovascularization and vascular leakage. We found that VEGFA (vascular endothelial growth factor A) stimulates Cttn phosphorylation at Y421, Y453, and Y470 residues in human retinal microvascular endothelial cells. In addition, we observed that while blockade of Cttn phosphorylation at Y470 inhibited VEGFA-induced human retinal microvascular endothelial cell angiogenic events, suppression of Y421 phosphorylation protected endothelial barrier integrity from disruption by VEGFA. In line with these observations, while blockade of Cttn phosphorylation at Y470 negated oxygen-induced retinopathy-induced retinal neovascularization, interference with Y421 phosphorylation prevented VEGFA/oxygen-induced retinopathy-induced vascular leakage. Mechanistically, while phosphorylation at Y470 was required for its interaction with Arp2/3 and CDC6 facilitating actin polymerization and DNA synthesis, respectively, Cttn phosphorylation at Y421 leads to its dissociation from VE-cadherin, resulting in adherens junction disruption. Furthermore, whereas Cttn phosphorylation at Y470 residue was dependent on Lyn, its phosphorylation at Y421 residue required Syk activation. Accordingly, lentivirus-mediated expression of shRNA targeting Lyn or Syk levels inhibited oxygen-induced retinopathy-induced retinal neovascularization and vascular leakage, respectively. The above observations show for the first time that phosphorylation of Cttn is involved in a site-specific manner in the regulation of retinal neovascularization and vascular leakage. In view of these findings, Cttn could be a novel target for the development of therapeutics against vascular diseases such as retinal neovascularization and vascular leakage.

Effects of Adipose Tissue-Specific Knockout of Delta-like Non-Canonical Notch Ligand 1 on Lipid Metabolism in Mice.

Delta-like non-canonical Notch ligand 1 (DLK1), which inhibits the differentiation of precursor adipocytes, is a recognized marker gene for precursor adipocytes. Lipids play a crucial role in energy storage and metabolism as a vital determinant of beef quality. In this study, we investigated the mechanism of the DLK1 gene in lipid metabolism by constructing adipose tissue-specific knockout mice. We examined some phenotypic traits, including body weight, liver coefficient, fat index, the content of triglyceride (TG) and cholesterol (CHOL) in abdominal white adipose tissue (WAT) and blood. Subsequently, the fatty acid content and genes related to lipid metabolism expression were detected in DLK1-/- and wild-type mice via GC-MS/MS analysis and quantitative real-time PCR (qRT-PCR), respectively. The results illustrated that DLK1-/- mice exhibited significant abdominal fat deposition compared to wild-type mice. HE staining and immunohistochemistry (IHC) results showed that the white adipocytes of DLK1-/- mice were larger, and the protein expression level of DLK1-/- was significantly lower. Regarding the blood biochemical parameters of female mice, DLK1-/- mice had a strikingly higher triglyceride content (p < 0.001). The fatty acid content in DLK1-/- mice was generally reduced. There was a significant reduction in the expression levels of the majority of genes that play a crucial role in lipid metabolism. This study reveals the molecular regulatory mechanism of fat metabolism in mice and provides a molecular basis and reference for the future application of the DLK1 gene in the breeding of beef cattle with an excellent meat quality traits. It also provides a molecular basis for unravelling the complex and subtle relationship between adipose tissue and health.

Hepatic farnesoid X receptor is necessary to facilitate ductular reaction and expression of heme biosynthetic genes.

Bile, which contains bile acids, the natural ligands for farnesoid x receptor (FXR), moves from the liver to the intestine through bile ducts. Ductular reaction often occurs during biliary obstruction. A subset of patients with erythropoietic protoporphyria, an inherited genetic mutation in heme biosynthetic enzyme ferrochelatase, accumulate porphyrin-containing bile plugs, leading to cholestasis. Here, we examined the link between FXR, bile plug formation, and how heme biosynthesis relates to this connection. We treated female and male wild-type and global and tissue-specific Fxr knockout mice with a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine, an inhibitor of ferrochelatase, and examined the expression of heme biosynthetic genes. We mined FXR mouse ChIP-Seq data, performed biochemical and histological analysis, and tested HepG2 and primary human hepatocytes after treatment with obeticholic acid, an FXR agonist. We observed that hepatic but not intestinal Fxr loss resulted in reduced bile plugs and ductular reaction in the liver. Then, we examined if FXR plays a regulatory role in heme biosynthesis and found significantly lower porphyrin accumulation in 3,5-diethoxycarbonyl-1, 4-dihydrocollidine-fed Fxr knockout mice. Gene expression and FXR mouse ChIP-Seq atlas analysis revealed that FXR orchestrates the expression of multiple heme biosynthetic enzymes. Finally, human HepG2 cells and primary human hepatocytes treated with obeticholic acid, showed increased expression of several heme biosynthetic genes. Overall, our data show that hepatic Fxr is necessary to maintain ductular reaction and accumulation of bile plugs. FXR can direct the expression of multiple heme biosynthetic genes. Thus, modulating FXR activity in EPP patients may help alleviate its associated liver disease.

Loss of ORP3 induces aneuploidy and promotes bladder cancer cell invasion through deregulated microtubule and actin dynamics

We have recently shown that loss of ORP3 leads to aneuploidy induction and promotes tumor formation. However, the specific mechanisms by which ORP3 contributes to ploidy-control and cancer initiation and progression is still unknown. Here, we report that ORP3 is highly expressed in ureter and bladder epithelium while its expression is downregulated in invasive bladder cancer cell lines and during tumor progression, both in human and in mouse bladder cancer. Moreover, we observed an increase in the incidence of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced invasive bladder carcinoma in the tissue-specific Orp3 knockout mice. Experimental data demonstrate that ORP3 protein interacts with γ-tubulin at the centrosomes and with components of actin cytoskeleton. Altering the expression of ORP3 induces aneuploidy and genomic instability in telomerase-immortalized urothelial cells with a stable karyotype and influences the migration and invasive capacity of bladder cancer cell lines. These findings demonstrate a crucial role of ORP3 in ploidy-control and indicate that ORP3 is a bona fide tumor suppressor protein. Of note, the presented data indicate that ORP3 affects both cell invasion and migration as well as genome stability through interactions with cytoskeletal components, providing a molecular link between aneuploidy and cell invasion and migration, two crucial characteristics of metastatic cells.

Loss of Ptpmt1 limits mitochondrial utilization of carbohydrates and leads to muscle atrophy and heart failure in tissue-specific knockout mice.

While mitochondria in different tissues have distinct preferences for energy sources, they are flexible in utilizing competing substrates for metabolism according to physiological and nutritional circumstances. However, the regulatory mechanisms and significance of metabolic flexibility are not completely understood. Here, we report that the deletion of Ptpmt1, a mitochondria-based phosphatase, critically alters mitochondrial fuel selection - the utilization of pyruvate, a key mitochondrial substrate derived from glucose (the major simple carbohydrate), is inhibited, whereas the fatty acid utilization is enhanced. Ptpmt1 knockout does not impact the development of the skeletal muscle or heart. However, the metabolic inflexibility ultimately leads to muscular atrophy, heart failure, and sudden death. Mechanistic analyses reveal that the prolonged substrate shift from carbohydrates to lipids causes oxidative stress and mitochondrial destruction, which in turn results in marked accumulation of lipids and profound damage in the knockout muscle cells and cardiomyocytes. Interestingly, Ptpmt1 deletion from the liver or adipose tissue does not generate any local or systemic defects. These findings suggest that Ptpmt1 plays an important role in maintaining mitochondrial flexibility and that their balanced utilization of carbohydrates and lipids is essential for both the skeletal muscle and the heart despite the two tissues having different preferred energy sources.

Loss of Ptpmt1 limits mitochondrial utilization of carbohydrates and leads to muscle atrophy and heart failure in tissue-specific knockout mice

While mitochondria in different tissues have distinct preferences for energy sources, they are flexible in utilizing competing substrates for metabolism according to physiological and nutritional circumstances. However, the regulatory mechanisms and significance of metabolic flexibility are not completely understood. Here, we report that the deletion of Ptpmt1, a mitochondria-based phosphatase, critically alters mitochondrial fuel selection – the utilization of pyruvate, a key mitochondrial substrate derived from glucose (the major simple carbohydrate), is inhibited, whereas the fatty acid utilization is enhanced. Ptpmt1 knockout does not impact the development of the skeletal muscle or heart. However, the metabolic inflexibility ultimately leads to muscular atrophy, heart failure, and sudden death. Mechanistic analyses reveal that the prolonged substrate shift from carbohydrates to lipids causes oxidative stress and mitochondrial destruction, which in turn results in marked accumulation of lipids and profound damage in the knockout muscle cells and cardiomyocytes. Interestingly, Ptpmt1 deletion from the liver or adipose tissue does not generate any local or systemic defects. These findings suggest that Ptpmt1 plays an important role in maintaining mitochondrial flexibility and that their balanced utilization of carbohydrates and lipids is essential for both the skeletal muscle and the heart despite the two tissues having different preferred energy sources.

Hepatic and intestinal manganese excretion are both required to regulate brain manganese during elevated manganese exposure.

Manganese (Mn) is essential, but neurotoxic at elevated levels. Under physiological conditions, Mn is primarily excreted by the liver, with the intestines playing a secondary role. Recent analyses of tissue-specific Slc30a10 or Slc39a14 knockout mice (SLC30A10 and SLC39A14 are Mn transporters) revealed that, under physiological conditions: (1) excretion of Mn by the liver and intestines is a major pathway that regulates brain Mn; and surprisingly, (2) the intestines compensate for loss of hepatic Mn excretion in controlling brain Mn. The unexpected importance of the intestines in controlling physiological brain Mn led us to determine the role of hepatic and intestinal Mn excretion in regulating brain Mn during elevated Mn exposure. We used liver- or intestine-specific Slc30a10 knockout mice as models to inhibit hepatic or intestinal Mn excretion. Compared with littermates, both knockout strains exhibited similar increases in brain Mn after elevated Mn exposure in early- or later-life. Thus, unlike physiological conditions, both hepatic and intestinal Mn excretion are required to control brain Mn during elevated Mn exposure. However, brain Mn levels of littermates and both knockout strains exposed to elevated Mn only in early-life were normalized in later-life. Thus, hepatic and intestinal Mn excretion play compensatory roles in clearing brain Mn accumulated by early-life Mn exposure. Finally, neuromotor assays provided evidence consistent with a role for hepatic and intestinal Mn excretion in functionally modulating Mn neurotoxicity during Mn exposure. Put together, these findings substantially enhance understanding of the regulation of brain Mn by excretion.

Deletion of hepatic growth hormone receptor (GHR) alters the mouse gut microbiota by affecting bile acid metabolism

ABSTRACT Both growth hormone (GH) and gut microbiota play significant roles in diverse physiological processes, but the crosstalk between them is poorly understood. Despite the regulation of GH by gut microbiota, study on GH’s influence on gut microbiota is limited, especially on the impacts of tissue specific GH signaling and their feedback effects on the host. In this study, we profiled gut microbiota and metabolome in tissue-specific GHR knockout mice in the liver (LKO) and adipose tissue (AKO). We found that GHR disruption in the liver rather than adipose tissue affected gut microbiota. It changed the abundance of Bacteroidota and Firmicutes at phylum level as well as abundance of several genera, such as Lactobacillus, Muribaculaceae, and Parasutterella, without affecting α-diversity. Moreover, the impaired liver bile acid (BA) profile in LKO mice was strongly associated with the change of gut microbiota. The BA pools and 12-OH BAs/non-12-OH BAs ratio were increased in the LKO mice, which was due to the induction of CYP8B1 by hepatic Ghr knockout. Consequently, the impaired BA pool in cecal content interacted with gut bacteria, which in turn increased the production of bacteria derived acetic acid, propionic acid, and phenylacetic acid that were possible to participate in the impaired metabolic phenotype of the LKO mice. Collectively, our findings suggested that the liver GH signaling regulates BA metabolism by its direct regulation on CYP8B1, which is an important factor influencing gut microbiota. Our study is significant in exploring gut microbiota modification effects of tissue-specific GH signaling as well as its involvement in gut microbiota–host interaction.

O-linked N-acetylglucosamine modification is essential for physiological adipose expansion induced by high-fat feeding.

Adipose tissues accumulate excess energy as fat and heavily influence metabolic homeostasis. O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation), which involves the addition of N-acetylglucosamine to proteins by O-GlcNAc transferase (Ogt), modulates multiple cellular processes. However, little is known about the role of O-GlcNAcylation in adipose tissues during body weight gain due to overnutrition. Here, we report on O-GlcNAcylation in mice with high-fat diet (HFD)-induced obesity. Mice with knockout of Ogt in adipose tissue achieved using adiponectin promoter-driven Cre recombinase (Ogt-FKO) gained less body weight than control mice under HFD. Surprisingly, Ogt-FKO mice exhibited glucose intolerance and insulin resistance, despite their reduced body weight gain, as well as decreased expression of de novo lipogenesis genes and increased expression of inflammatory genes, resulting in fibrosis at 24 weeks of age. Primary cultured adipocytes derived from Ogt-FKO mice showed decreased lipid accumulation. Both primary cultured adipocytes and 3T3-L1 adipocytes treated with OGT inhibitor showed increased secretion of free fatty acids. Medium derived from these adipocytes stimulated inflammatory genes in RAW 264.7 macrophages, suggesting that cell-to-cell communication via free fatty acids might be a cause of adipose inflammation in Ogt-FKO mice. In conclusion, O-GlcNAcylation is important for healthy adipose expansion in mice. Glucose flux into adipose tissues may be a signal to store excess energy as fat.NEW & NOTEWORTHY We evaluated the role of O-GlcNAcylation in adipose tissue in diet-induced obesity using adipose tissue-specific Ogt knockout mice. We found that O-GlcNAcylation in adipose tissue is essential for healthy fat expansion and that Ogt-FKO mice exhibit severe fibrosis upon long-term overnutrition. O-GlcNAcylation in adipose tissue may regulate de novo lipogenesis and free fatty acid efflux to the degree of overnutrition. We believe that these results provide new insights into adipose tissue physiology and obesity research.

PO-01-147 ACTIVATION OF AUTOPHAGY ATTENUATES PROGRESSION OF ARRHYTHMOGENIC RIGHT VENTRICULAR CARDIOMYOPATHY

Studies of autophagy in ARVC are quite rare. Even autophagy had the opposite effects in desmin-related cardiomyopathy and dilated cardiomyopathy. This study aimed to explore the regulatory role of autophagy in the progression of ARVC. The ARVC cardiomyocytes (HL-1/shJup) were treated with autophagy activator (rapamycin) and inhibitor (chloroquine) to manipulate autophagy activity. Western blotting and immunofluorescence determined the changes of disease progression and signaling pathways. Then, autophagy was manipulated in cardiac tissue-specific Jup knockout mice to confirm the cell-derived findings. The autophagy activity of HL-1/shJup cell line was higher than that of control cells. In HL-1/shJup cells, the upstream signaling pathway of autophagy was active. During rapamycin treatment, the proteins related to lipogenesis and fibrogenesis in HL-1/shJup cells were reduced. In contrast, chloroquine treatment had the opposite effect. Moreover, rapamycin decreased and chloroquine increased intracellular oil droplet accumulation in HL-1/shJup cells. In addition, there was no significant change in Wnt/Hippo signaling pathways in cardiomyocytes after rapamycin treatment. Instead, the protein expression of the transcription factor SREBP1 that regulates lipogenesis and lysyl oxidase (LOX) that promotes fibrosis were related to the regulatory mechanism of autophagy. In ARVC mice, activated/inhibited autophagy also reduced/increased RV distance, arrhythmia, and lipogenesis through SREBP1, but not in wild-type mice. Autophagy plays a protective role in ARVC by regulating lipogenesis to attenuate disease progression. Based on this study, autophagy activators may be used in combination with existing drugs in patients with ARVC disease in the future to provide additional protection for early heart failure or heart transplantation.

Abstract 5919: The significant role of HMGB1 in mesothelial cells in contributing to the mesothelioma development induced by asbestos

Abstract Diffuse Malignant mesothelioma is a very aggressive cancer that is related to asbestos exposure. We found previously that following asbestos deposition in the pleura and in the peritoneum, mesothelial cells release HMGB1 that attract granulocytes and macrophages that, in turn, secrete HMGB1. It was unknown whether the primary source of HMGB1 that drives the inflammatory process caused by asbestos, which may ensue in mesothelioma, derives from the mesothelial cells exposed to asbestos or from the granulocytes and macrophages. To study separately the contribution of mesothelial cells and of the inflammatory cells to HMGB1 secretion that drives mesothelioma development, we used two tissue-specific HMGB1 knockout mouse models: (1) An inducible mesothelial conditional HMGB1 knockout (HMGB1ΔpMeso) mouse model, and (2) a constitutive myelomonocytic-lineage knock out (HMGB1ΔMylc) mouse model. To investigate the role of HMGB1 in asbestos induced carcinogenesis in vivo, we conducted both short-term and long-term experiments in HMGB1ΔpMeso mice and in HMGB1ΔMylc mice. For the Short-term experiment, we observed that one week following the completion of the 10-weekly crocidolite i.p. injections, all the mice in experimental and control groups showed inflammation with multiple granulomas around asbestos deposits in the various tissues and organs present in the abdomen, and diffuse mesothelial hyperplasia. However, the granulomas in HMGB1ΔpMeso mice were significantly smaller compared to those found in the control WT mice. TNFα IHC stain was minimal to non-detectable in the granulomas and areas of mesothelial hyperplasia of HMGB1ΔpMeso mice, compared to a strong TNFα staining in both HMGB1ΔMylc and WT mice. For the Long-term experiment, we observed that the incidence of mesothelioma was significantly lower in HMGB1ΔpMeso mice compared with the 3 control groups including WT mice, HMGB1F/F mice and Wt1ERT2Cre/+ control mice. Moreover, HMGB1ΔpMeso mice had a significantly longer mesothelioma-specific survival compared to all three controls. Histologically, the HMGB1ΔpMeso mesotheliomas were smaller and grew largely over the surface rather than infiltrating the abdominal organs, compared to the mesotheliomas that developed in the three control groups. In summary, our findings underscore the role of HMGB1 in asbestos pathogenesis, including asbestos induced chronic inflammation and mesothelioma. Moreover, our findings elucidated the mechanisms responsible for asbestos induced inflammation and asbestos carcinogenesis, and the causal link between HMGB1, TNFα secretion and mesothelioma. Our results point to the HMGB1 secreted by mesothelial cells as the culprit and thus the ideal target to prevent the growth of mesothelioma upon asbestos exposure and during the early phases of mesothelioma growth. Citation Format: Joelle S. Suarez, Giovanni Gaudino, Tak W. Mak, Michele Carbone, Haining Yang. The significant role of HMGB1 in mesothelial cells in contributing to the mesothelioma development induced by asbestos [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5919.

Generation of Brown Fat-Specific Knockout Mice Using a Combined Cre-LoxP, CRISPR-Cas9, and Adeno-Associated Virus Single-Guide RNA System.

Brown adipose tissue (BAT) is an adipose depot specialized in energy dissipation that can also serve as an endocrine organ via the secretion of bioactive molecules. The creation of BAT-specific knockout mice is one of the most popular approaches for understanding the contribution of a gene of interest to BAT-mediated energy regulation. The conventional gene targeting strategy utilizing the Cre-LoxP system has been the principal approach to generate tissue-specific knockout mice. However, this approach is time-consuming and tedious. Here, we describe a protocol for the rapid and efficient knockout of a gene of interest in BAT using a combined Cre-LoxP, CRISPR-Cas9, and adeno-associated virus (AAV) single-guide RNA (sgRNA) system. The interscapular BAT is located in the deep layer between the muscles. Thus, the BAT must be exposed in order to inject the AAV precisely and directly into the BAT within the visual field. Appropriate surgical handling is crucial to prevent damage to the sympathetic nerves and vessels, such as the Sultzer's vein that connects to the BAT. To minimize tissue damage, there is a critical need to understand the three-dimensional anatomical location of the BAT and the surgical skills required in the technical steps. This protocol highlights the key technical procedures, including the design of sgRNAs targeting the gene of interest, the preparation of AAV-sgRNA particles, and the surgery for the direct microinjection of AAV into both BAT lobes for generating BAT-specific knockout mice, which can be broadly applied to study the biological functions of genes in BAT.

SIRT7 suppresses energy expenditure and thermogenesis by regulating brown adipose tissue functions in mice

Brown adipose tissue plays a central role in the regulation of the energy balance by expending energy to produce heat. NAD+-dependent deacylase sirtuins have widely been recognized as positive regulators of brown adipose tissue thermogenesis. However, here we reveal that SIRT7, one of seven mammalian sirtuins, suppresses energy expenditure and thermogenesis by regulating brown adipose tissue functions. Whole-body and brown adipose tissue-specific Sirt7 knockout mice have higher body temperature and energy expenditure. SIRT7 deficiency increases the protein level of UCP1, a key regulator of brown adipose tissue thermogenesis. Mechanistically, we found that SIRT7 deacetylates insulin-like growth factor 2 mRNA-binding protein 2, an RNA-binding protein that inhibits the translation of Ucp1 mRNA, thereby enhancing its inhibitory action on Ucp1. Furthermore, SIRT7 attenuates the expression of batokine genes, such as fibroblast growth factor 21. In conclusion, we propose that SIRT7 serves as an energy-saving factor by suppressing brown adipose tissue functions.

RNA binding protein HuR protects against NAFLD by suppressing long noncoding RNA H19 expression

BackgroundNAFLD has become the most common chronic liver disease worldwide. Human antigen R (HuR), an RNA-binding protein, is an important post-transcriptional regulator. HuR has been reported as a key player in regulating lipid homeostasis in the liver and adipose tissues by using tissue-specific HuR knockout mice. However, the underlying mechanism by which hepatocyte-specific HuR regulates hepatic lipid metabolism under metabolic stress remains unclear and is the focus of this study.MethodsHepatocyte-specific HuR deficient mice (HuRhKO) and age-/gender-matched control mice, as well as long-noncoding RNA H19 knockout mice (H19−/−), were fed a Western Diet plus sugar water (WDSW). Hepatic lipid accumulation, inflammation and fibrosis were examined by histology, RNA transcriptome analysis, qRT–PCR, and Western blot analysis. Bile acid composition was measured using LC–MS/MS.ResultsHepatocyte-specific deletion of HuR not only significantly increased hepatic lipid accumulation by modulating fatty acid synthesis and metabolism but also markedly induced inflammation by increasing immune cell infiltration and neutrophil activation under metabolic stress. In addition, hepatic deficiency of HuR disrupted bile acid homeostasis and enhanced liver fibrosis. Mechanistically, HuR is a repressor of H19 expression. Analysis of a recently published dataset (GSE143358) identified H19 as the top-upregulated gene in liver-specific HuR knockout mice. Similarly, hepatocyte-specific deficiency of HuR dramatically induced the expression of H19 and sphingosine-1 phosphate receptor 2 (S1PR2), but reduced the expression of sphingosine kinase 2 (SphK2). WDSW-induced hepatic lipid accumulation was alleviated in H19−/− mice. Furthermore, the downregulation of H19 alleviated WDSW-induced NAFLD in HuRhKO mice.ConclusionsHuR not only functions as an RNA binding protein to modulate post-transcriptional gene expression but also regulates H19 promoter activity. Hepatic HuR is an important regulator of hepatic lipid metabolism via modulating H19 expression.