Glucose regulation of adipose tissue browning by CBP/p300- and HDAC3-mediated reversible acetylation of CREBZF.

Glucose is required for generating heat during cold-induced nonshivering thermogenesis in adipose tissue, but the regulatory mechanism is largely unknown. CREBZF has emerged as a critical mechanism for metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease (NAFLD). We investigated the roles of CREBZF in the control of thermogenesis and energy metabolism. Glucose induces CREBZF in human white adipose tissue (WAT) and inguinal WAT (iWAT) in mice. Lys208 acetylation modulated by transacetylase CREB-binding protein/p300 and deacetylase HDAC3 is required for glucose-induced reduction of proteasomal degradation and augmentation of protein stability of CREBZF. Glucose induces rectal temperature and thermogenesis in white adipose of control mice, which is further potentiated in adipose-specific CREBZF knockout (CREBZF FKO) mice. During cold exposure, CREBZF FKO mice display enhanced thermogenic gene expression, browning of iWAT, and adaptive thermogenesis. CREBZF associates with PGC-1α to repress thermogenic gene expression. Expression levels of CREBZF are negatively correlated with UCP1 in human adipose tissues and increased in WAT of obese ob/ob mice, which may underscore the potential role of CREBZF in the development of compromised thermogenic capability under hyperglycemic conditions. Our results reveal an important mechanism of glucose sensing and thermogenic inactivation through reversible acetylation.

Antibacterial activity of isopropoxy benzene guanidine against Riemerella anatipestifer.

Introduction: Riemerella anatipestifer (R. anatipestifer) is an important pathogen in waterfowl, leading to substantial economic losses. In recent years, there has been a notable escalation in the drug resistance rate of R. anatipestifer. Consequently, there is an imperative need to expedite the development of novel antibacterial medications to effectively manage the infection caused by R. anatipestifer. Methods: This study investigated the in vitro and in vivo antibacterial activities of a novel substituted benzene guanidine analog, namely, isopropoxy benzene guanidine (IBG), against R. anatipestifer by using the microdilution method, time-killing curve, and a pericarditis model. The possible mechanisms of these activities were explored. Results and Discussion: The minimal inhibitory concentration (MIC) range of IBG for R. anatipestifer was 0.5-2 µg/mL. Time-killing curves showed a concentration-dependent antibacterial effect. IBG alone or in combination with gentamicin significantly reduced the bacterial load of R. anatipestifer in the pericarditis model. Serial-passage mutagenicity assays showed a low probability for developing IBG resistance. Mechanistic studies suggested that IBG induced membrane damage by binding to phosphatidylglycerol and cardiolipin, leading to an imbalance in membrane potential and the transmembrane proton gradient, as well as the decreased of intracellular adenosine triphosphate. In summary, IBG is a potential antibacterial for controlling R. anatipestifer infections.

Monovalent vaccination with inactivated SARS-CoV-2 BA.5 protects hamsters against Omicron but not non-Omicron variants.

We compared the protective effects of inactivated SARS-CoV-2 vaccines derived from the ancestral and the currently circulating BA.5.2 strains against infection with multiple variants in Syrian golden hamsters. Vaccination with BA.5.2 effectively protected against infection with the Omicron subvariants including XBB.1, but not the Alpha or Delta variant. In contrast, hamsters vaccinated with the ancestral strain demonstrated decent neutralization activity against both the Omicron and non-Omicron variants. Our findings might instruct future design and formulation of SARS-CoV-2 vaccines.

Proline hydroxylation of CREB-regulated transcriptional coactivator 2 controls hepatic glucose metabolism.

Prolyl hydroxylase domain (PHD) enzymes change HIF activity according to oxygen signal; whether it is regulated by other physiological conditions remains largely unknown. Here, we report that PHD3 is induced by fasting and regulates hepatic gluconeogenesis through interaction and hydroxylation of CRTC2. Pro129 and Pro615 hydroxylation of CRTC2 following PHD3 activation is necessary for its association with cAMP-response element binding protein (CREB) and nuclear translocation, and enhanced binding to promoters of gluconeogenic genes by fasting or forskolin. CRTC2 hydroxylation-stimulated gluconeogenic gene expression is independent of SIK-mediated phosphorylation of CRTC2. Liver-specific knockout of PHD3 (PHD3 LKO) or prolyl hydroxylase-deficient knockin mice (PHD3 KI) show attenuated fasting gluconeogenic genes, glycemia, and hepatic capacity to produce glucose during fasting or fed with high-fat, high-sucrose diet. Importantly, Pro615 hydroxylation of CRTC2 by PHD3 is increased in livers of fasted mice, diet-induced insulin resistance or genetically obese ob/ob mice, and humans with diabetes. These findings increase our understanding of molecular mechanisms linking protein hydroxylation to gluconeogenesis and may offer therapeutic potential for treating excessive gluconeogenesis, hyperglycemia, and type 2 diabetes.

Intrahepatic osteopontin signaling by CREBZF defines a checkpoint for steatosis-to-NASH progression.

BACKGROUND AND AIMS: NASH has emerged as a leading cause of chronic liver disease. However, the mechanisms that govern NASH fibrosis remain largely unknown. CREBZF is a CREB/ATF bZIP transcription factor that causes hepatic steatosis and metabolic defects in obesity. APPROACH AND RESULTS: Here, we show that CREBZF is a key mechanism of liver fibrosis checkpoint that promotes hepatocyte injury and exacerbates diet-induced NASH in mice. CREBZF deficiency attenuated liver injury, fibrosis, and inflammation in diet-induced mouse models of NASH. CREBZF increases HSC activation and fibrosis in a hepatocyte-autonomous manner by stimulating an extracellular matrix protein osteopontin, a key regulator of fibrosis. The inhibition of miR-6964-3p mediates CREBZF-induced production and secretion of osteopontin in hepatocytes. Adeno-associated virus -mediated rescue of osteopontin restored HSC activation, liver fibrosis, and NASH progression in CREBZF-deficient mice. Importantly, expression levels of CREBZF are increased in livers of diet-induced NASH mouse models and humans with NASH. CONCLUSIONS: Osteopontin signaling by CREBZF represents a previously unrecognized intrahepatic mechanism that triggers liver fibrosis and contributes to the severity of NASH.

Sodium Butyrate Supplementation Inhibits Hepatic Steatosis by Stimulating Liver Kinase B1 and Insulin-Induced Gene.

BACKGROUND AND AIMS: Butyric acid is an intestinal microbiota-produced short-chain fatty acid, which exerts salutary effects on alleviating nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanism of butyrate on regulating hepatic lipid metabolism is largely unexplored. METHODS: A mouse model of NAFLD was induced with high-fat diet feeding, and sodium butyrate (NaB) intervention was initiated at the eighth week and lasted for 8 weeks. Hepatic steatosis was evaluated and metabolic pathways concerning lipid homeostasis were analyzed. RESULTS: Here, we report that administration of NaB by gavage once daily for 8 weeks causes an augmentation of insulin-induced gene (Insig) activity and inhibition of lipogenic gene in mice fed with high-fat diet. Mechanistically, NaB is sufficient to enhance the interaction between Insig and its upstream kinase AMP-activated protein kinase (AMPK). The stimulatory effects of NaB on Insig-1 activity are abolished in AMPKα1/α2 double knockout (AMPK-/-) mouse primary hepatocytes. Moreover, AMPK activation by NaB is mediated by LKB1, as evidenced by the observations showing NaB-mediated induction of phosphorylation of AMPK, and its downstream target acetyl-CoA carboxylase is diminished in LKB1-/- mouse embryonic fibroblasts. CONCLUSIONS: These studies indicate that NaB serves as a negative regulator of hepatic lipogenesis in NAFLD and that NaB attenuates hepatic steatosis and improves lipid profile and liver function largely through the activation of LKB1-AMPK-Insig signaling pathway. Therefore, NaB has therapeutic potential for treating NAFLD and related metabolic diseases.

Thrombospondin 1 improves hepatic steatosis in diet-induced insulin-resistant mice and is associated with hepatic fat content in humans.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is associated with altered production of secreted proteins. Increased understanding of secreted proteins could lead to improved prediction and treatment of NAFLD. Here, we aimed to discover novel secreted proteins in humans that are associated with hepatic fat content using unbiased proteomic profiling strategy, and how the identified Thbs1 modulates lipid metabolism and hepatic steatosis. METHOD: NAFLD patients were enrolled and treated with lifestyle intervention. Patients who underwent liver biopsy were enrolled for analyzing the correlation between circulating Thbs1 and liver steatosis. Mice were fed on high-fat, high-sucrose diet and treated with recombinant Thbs1. Primary hepatocytes isolated from CD36 knockout (CD36-/-) mice and their wild-type littermates (controls) were treated with glucose plus insulin for 24 h together with or without recombinant Thbs1. FINDING: Serum Thbs1 levels are increased in participants with NAFLD and positively associated with liver steatosis grades. Improvement of liver steatosis after lifestyle intervention was accompanied with significant reduction of serum Thbs1 levels. Pharmacological administration of recombinant human Thbs1 attenuates hepatic steatosis in diet-induced obese mice. Treatment with Thbs1 protein or stably overexpression of Thbs1 causes a significant reduction of lipid accumulation in primary hepatocytes or HepG2 cells exposed to high glucose plus insulin, suggesting that Thbs1 regulates lipid metabolism in a hepatocyte-autonomous manner. Mechanistically, Thbs1 inhibits cleavage and processing of SREBP-1, leading to a reduction of target lipogenic gene expression and hepatic steatosis. Inhibitory effects of Thbs1 on lipogenesis and triglyceride accumulation are abrogated in CD36 deficient primary hepatocytes exposed to high glucose plus insulin. Interestingly, beneficial effects of Thbs1 on lipid accumulation are observed in primary hepatocytes treated with a Thbs1 nonapeptide mimetic ABT-526. INTERPRETATION: Thbs1 is a biomarker for NAFLD in humans, and pharmacological and genetic approaches for the modulation of Thbs1 activity may have the therapeutic potential for treating hepatic steatosis. FUND: A full list of funding bodies that contributed to this study can be found in the Funding Sources section.

The Effects of B1344, a Novel Fibroblast Growth Factor 21 Analog, on Nonalcoholic Steatohepatitis in Nonhuman Primates.

Nonalcoholic steatohepatitis has emerged as a major cause of liver diseases with no effective therapies. Here, we evaluate the efficacies and pharmacokinetics of B1344, a long-acting polyethylene glycolylated (PEGylated) fibroblast growth factor 21 analog, in a nongenetically modified nonhuman primate species that underwent liver biopsy and demonstrate the potential for efficacies in humans. B1344 is sufficient to selectively activate signaling from the ßKlotho/FGFR1c receptor complex. In cynomolgus monkeys with nonalcoholic fatty liver disease (NAFLD), administration of B1344 via subcutaneous injection for 11 weeks caused a profound reduction of hepatic steatosis, inflammation, and fibrosis, along with amelioration of liver injury and hepatocyte death, as evidenced by liver biopsy specimen and biochemical analysis. Moreover, improvement of metabolic parameters was observed in the monkeys, including reduction of body weight and improvement of lipid profiles and glycemic control. To determine the role of B1344 in the progression of murine NAFLD independent of obesity, B1344 was administered to mice fed a methionine- and choline-deficient diet. Consistently, B1344 administration prevented the mice from lipotoxicity damage and nonalcoholic steatohepatitis in a dose-dependent manner. These results provide preclinical validation for an innovative therapeutic approach to NAFLD and support further clinical testing of B1344 for treating nonalcoholic steatohepatitis and other metabolic diseases in humans.

CREBZF as a Key Regulator of STAT3 Pathway in the Control of Liver Regeneration in Mice.

BACKGROUND AND AIMS: STAT3, a member of the signal transducer and activator of transcription (STAT) family, is strongly associated with liver injury, inflammation, regeneration, and hepatocellular carcinoma development. However, the signals that regulate STAT3 activity are not completely understood. APPROACH AND RESULTS: Here we characterize CREB/ATF bZIP transcription factor CREBZF as a critical regulator of STAT3 in the hepatocyte to repress liver regeneration. We show that CREBZF deficiency stimulates the expression of the cyclin gene family and enhances liver regeneration after partial hepatectomy. Flow cytometry analysis reveals that CREBZF regulates cell cycle progression during liver regeneration in a hepatocyte-autonomous manner. Similar results were observed in another model of liver regeneration induced by intraperitoneal injection of carbon tetrachloride (CCl4 ). Mechanistically, CREBZF potently associates with the linker domain of STAT3 and represses its dimerization and transcriptional activity in vivo and in vitro. Importantly, hepatectomy-induced hyperactivation of cyclin D1 and liver regeneration in CREBZF liver-specific knockout mice was reversed by selective STAT3 inhibitor cucurbitacin I. In contrast, adeno-associated virus-mediated overexpression of CREBZF in the liver inhibits the expression of the cyclin gene family and attenuates liver regeneration in CCl4 -treated mice. CONCLUSIONS: These results characterize CREBZF as a coregulator of STAT3 to inhibit regenerative capacity, which may represent an essential cellular signal to maintain liver mass homeostasis. Therapeutic approaches to inhibit CREBZF may benefit the compromised liver during liver transplantation.

Indole-3-propionic acid inhibits gut dysbiosis and endotoxin leakage to attenuate steatohepatitis in rats.

Microbial metabolites have emerged as critical components that mediate the metabolic effects of the gut microbiota. Here, we show that indole-3-propionic acid (IPA), a tryptophan metabolite produced by gut bacteria, is a potent anti-non-alcoholic steatohepatitis (NASH) microbial metabolite. Here, we demonstrate that administration of IPA modulates the microbiota composition in the gut and inhibits microbial dysbiosis in rats fed a high-fat diet. IPA induces the expression of tight junction proteins, such as ZO-1 and Occludin, and maintains intestinal epithelium homeostasis, leading to a reduction in plasma endotoxin levels. Interestingly, IPA inhibits NF-κB signaling and reduces the levels of proinflammatory cytokines, such as TNFα, IL-1ß, and IL-6, in response to endotoxin in macrophages to repress hepatic inflammation and liver injury. Moreover, IPA is sufficient to inhibit the expression of fibrogenic and collagen genes and attenuate diet-induced NASH phenotypes. The beneficial effects of IPA on the liver are likely mediated through inhibiting the production of endotoxin in the gut. These findings suggest a protective role of IPA in the control of metabolism and uncover the gut microbiome and liver cross-talk in regulating the intestinal microenvironment and liver pathology via a novel dietary nutrient metabolite. IPA may provide a new therapeutic strategy for treating NASH.

Trimethylamine N-oxide attenuates high-fat high-cholesterol diet-induced steatohepatitis by reducing hepatic cholesterol overload in rats.

BACKGROUND: Trimethylamine N-oxide (TMAO) has been shown to be involved in cardiovascular disease (CVD). However, its role in nonalcoholic steatohepatitis (NASH) is unknown. AIM: To determine the effect of TMAO on the progression of NASH. METHODS: A rat model was induced by 16-wk high-fat high-cholesterol (HFHC) diet feeding and TMAO was administrated by daily oral gavage for 8 wk. RESULTS: Oral TMAO intervention attenuated HFHC diet-induced steatohepatitis in rats. Histological evaluation showed that TMAO treatment significantly alleviated lobular inflammation and hepatocyte ballooning in the livers of rats fed a HFHC diet. Serum levels of alanine aminotransferase and aspartate aminotransferase were also decreased by TMAO treatment. Moreover, hepatic endoplasmic reticulum (ER) stress and cell death were mitigated in HFHC diet-fed TMAO-treated rats. Hepatic and serum levels of cholesterol were both decreased by TMAO treatment in rats fed a HFHC diet. Furthermore, the expression levels of intestinal cholesterol transporters were detected. Interestingly, cholesterol influx-related Niemann-Pick C1-like 1 was downregulated and cholesterol efflux-related ABCG5/8 were upregulated by TMAO treatment in the small intestine. Gut microbiota analysis showed that TMAO could alter the gut microbial profile and restore the diversity of gut flora. CONCLUSION: These data suggest that TMAO may modulate the gut microbiota, inhibit intestinal cholesterol absorption, and ameliorate hepatic ER stress and cell death under cholesterol overload, thereby attenuating HFHC diet-induced steatohepatitis in rats. Further studies are needed to evaluate the influence on CVD and define the safe does of TMAO treatment.

Prolyl hydroxylase 3 stabilizes the p53 tumor suppressor by inhibiting the p53-MDM2 interaction in a hydroxylase-independent manner.

Prolyl hydroxylase 3 (PHD3) has initially been reported to hydroxylase hypoxia-inducible factor α (HIFα) and mediate HIFα degradation. More recent studies have shown that, in addition to HIFα, PHD3 has also other substrates. Moreover, pHD3 is believed to act as a tumor suppressor, but the underlying mechanism remains to be elucidated. Here, we demonstrate that PHD3 stabilizes p53 in a hydroxylase-independent manner. We found that PHD3 overexpression increases and PHD3 knockdown decreases p53 levels. Mechanistically, PHD3 bound MDM2 proto-oncogene (MDM2) and prevented MDM2 from interacting with p53, thereby inhibiting MDM2-mediated p53 degradation. Interestingly, we found that PHD3 overexpression could enhance p53 in the presence of the prolyl hydroxylase inhibitor dimethyloxalylglycine, and the prolyl hydroxylase activity-deficient variant PHD3-H196A also inhibited the p53-MDM2 interaction and stabilized p53. Genetic ablation of PHD3 decreased p53 protein levels in mice intestinal epithelial cells, but a genetic knockin of PHD3-H196A did not affect p53 protein levels in vivo These results suggest that the prolyl hydroxylase activity of PHD3 is dispensable for its ability to stabilize p53. We found that both PHD3 and PHD3-H196A suppress the expression of the stem cell-associated gene NANOG and inhibited the properties of colon cancer stem cells through p53. Our results reveal an additional critical mechanism underlying the regulation of p53 expression and highlight that PHD3 plays a role in the suppression of colon cancer cell stemness in a hydroxylase-independent manner.

Post-translational regulation of lipogenesis via AMPK-dependent phosphorylation of insulin-induced gene.

Insulin-induced gene (Insig) negatively regulates SREBP-mediated de novo fatty acid synthesis in the liver. However, the upstream regulation of Insig is incompletely understood. Here we report that AMPK interacts with and mediates phosphorylation of Insig. Thr222 phosphorylation following AMPK activation is required for protein stabilization of Insig-1, inhibition of cleavage and processing of SREBP-1, and lipogenic gene expression in response to metformin or A769662. AMPK-dependent phosphorylation ablates Insig's interaction with E3 ubiquitin ligase gp78 and represses its ubiquitination and degradation, whereas AMPK deficiency shows opposite effects. Interestingly, activation of AMPK by metformin causes an augmentation of Insig stability and reduction of lipogenic gene expression, and leads to the attenuation of hepatic steatosis in HFHS diet-fed mice. Moreover, hepatic overexpression of Insig-1 rescues hepatic steatosis in liver-specific AMPKα2 knockout mice fed with HFHS diet. These findings uncover a novel effector of AMPK. Targeting Insig may have the therapeutic potential for treating fatty liver disease and related disorders.

Hepatic CREBZF couples insulin to lipogenesis by inhibiting insig activity and contributes to hepatic steatosis in diet-induced insulin-resistant mice.

Insulin is critical for the regulation of de novo fatty acid synthesis, which converts glucose to lipid in the liver. However, how insulin signals are transduced into the cell and then regulate lipogenesis remains to be fully understood. Here, we identified CREB/ATF bZIP transcription factor (CREBZF) of the activating transcription factor/cAMP response element-binding protein (ATF/CREB) gene family as a key regulator for lipogenesis through insulin-Akt signaling. Insulin-induced gene 2a (Insig-2a) decreases during refeeding, allowing sterol regulatory element binding protein 1c to be processed to promote lipogenesis; but the mechanism of reduction is unknown. We show that Insig-2a inhibition is mediated by insulin-induced CREBZF. CREBZF directly inhibits transcription of Insig-2a through association with activating transcription factor 4. Liver-specific knockout of CREBZF causes an induction of Insig-2a and Insig-1 and resulted in repressed lipogenic program in the liver of mice during refeeding or upon treatment with streptozotocin and insulin. Moreover, hepatic CREBZF deficiency attenuates hepatic steatosis in high-fat, high-sucrose diet-fed mice. Importantly, expression levels of CREBZF are increased in livers of diet-induced insulin resistance or genetically obese ob/ob mice and humans with hepatic steatosis, which may underscore the potential role of CREBZF in the development of sustained lipogenesis in the liver under selective insulin resistance conditions. CONCLUSION: These findings uncover an unexpected mechanism that couples changes in extracellular hormonal signals to hepatic lipid homeostasis; disrupting CREBZF function may have the therapeutic potential for treating fatty liver disease and insulin resistance. (Hepatology 2018).