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.

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.

Aging exaggerates acute-on-chronic alcohol-induced liver injury in mice and humans by inhibiting neutrophilic sirtuin 1-C/EBPα-miRNA-223 axis.

BACKGROUND AND AIMS: Aging exacerbates liver neutrophil infiltration and alcohol-associated liver disease (ALD) in mice and humans, but the underlying mechanisms remain obscure. This study aimed to examine the effect of aging and alcohol consumption on neutrophilic Sirtuin 1 (SIRT1) and microRNA-223 (miR-223), and their contribution to ALD pathogeneses. APPROACH AND RESULTS: Young and aged myeloid-specific Sirt1 knockout mice were subjected to chronic-plus-binge ethanol feeding. Blood samples from healthy controls and patients with chronic alcohol drinking who presented with acute intoxication were analyzed. Neutrophilic Sirt1 and miR-223 expression were down-regulated in aged mice compared with young mice. Deletion of the Sirt1 gene in myeloid cells including neutrophils exacerbated chronic-plus-binge ethanol-induced liver injury and inflammation and down-regulated neutrophilic miR-223 expression. Immunoprecipitation experiments revealed that SIRT1 promoted C/EBPα deacetylation by directly interacting with C/EBPα, a key transcription factor that controls miR-223 biogenesis, and subsequently elevated miR-223 expression in neutrophils. Importantly, down-regulation of SIRT1 and miR-223 expression was also observed in circulating neutrophils from middle-aged and elderly subjects compared with those from young individuals. Chronic alcohol users with acute intoxication had a reduction in neutrophilic SIRT1 expression in young and middle-aged patients, with a greater reduction in the latter group. The neutrophilic SIRT1 expression correlated with neutrophilic miR-223 and serum alanine transaminase levels in those patients. CONCLUSIONS: Aging increases the susceptibility of alcohol-induced liver injury in mice and humans through the down-regulation of the neutrophilic SIRT1-C/EBPα-miR-223 axis, which could be a therapeutic target for the prevention and/or treatment of ALD.

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.

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).

Fibroblast growth factor 21 improves hepatic insulin sensitivity by inhibiting mammalian target of rapamycin complex 1 in mice.

UNLABELLED: Among the 22 fibroblast growth factors (FGFs), FGF21 has now emerged as a key metabolic regulator. However, the mechanism whereby FGF21 mediates its metabolic actions per se remains largely unknown. Here, we show that FGF21 represses mammalian target of rapamycin complex 1 (mTORC1) and improves insulin sensitivity and glycogen storage in a hepatocyte-autonomous manner. Administration of FGF21 in mice inhibits mTORC1 in the liver, whereas FGF21-deficient mice display pronounced insulin-stimulated mTORC1 activation and exacerbated hepatic insulin resistance (IR). FGF21 inhibits insulin- or nutrient-stimulated activation of mTORC1 to enhance phosphorylation of Akt in HepG2 cells at both normal and IR condition. TSC1 deficiency abrogates FGF21-mediated inhibition of mTORC1 and augmentation of insulin signaling and glycogen synthesis. Strikingly, hepatic ßKlotho knockdown or hepatic hyperactivation of mTORC1/ribosomal protein S6 kinase 1 abrogates hepatic insulin-sensitizing and glycemic-control effects of FGF21 in diet-induced insulin-resistant mice. Moreover, FGF21 improves methionine- and choline-deficient diet-induced steatohepatitis. CONCLUSIONS: FGF21 acts as an inhibitor of mTORC1 to control hepatic insulin action and maintain glucose homeostasis, and mTORC1 inhibition by FGF21 has the therapeutic potential for treating IR and type 2 diabetes. (Hepatology 2016;64:425-438).

Identification of BAF60b as a Chromatin-Remodeling Checkpoint of Diet-Induced Fatty Liver Disease.

Overnutrition has gradually become the primary causative factor in nonalcoholic fatty liver disease (NAFLD). However, how nutritional signals are integrated to orchestrate the transcriptional programs important for NAFLD progression remains poorly understood. We identified hepatic BAF60b as a lipid-sensitive subunit of the switch/sucrose nonfermentable chromatin-remodeling complex that is negatively associated with liver steatosis in mice and humans. Hepatic BAF60b deficiency promotes high-fat diet (HFD)-induced liver steatosis in mice, whereas transgenic expression of BAF60b in the liver attenuates HFD-induced obesity and NAFLD, both accompanied by a marked regulation of peroxisome proliferator-activated receptor γ (PPARγ) expression. Mechanistically, through motif analysis of liver assay for transposase-accessible chromatin sequencing and multiple validation experiments, we identified C/EBPß as the transcription factor that interacts with BAF60b to suppress Pparγ gene expression, thereby controlling hepatic lipid accumulation and NAFLD progression. This work identifies hepatic BAF60b as a negative regulator of liver steatosis through C/EBPß-dependent chromatin remodeling.

Macrophage CREBZF Orchestrates Inflammatory Response to Potentiate Insulin Resistance and Type 2 Diabetes.

Chronic adipose tissue inflammation accompanied by macrophage accumulation and activation is implicated in the pathogenesis of insulin resistance and type 2 diabetes in humans. The transcriptional coregulator CREBZF is a key factor in hepatic metabolism, yet its role in modulating adipose tissue inflammation and type 2 diabetes remains elusive. The present study demonstrates that overnutrition-induced CREBZF links adipose tissue macrophage (ATM) proinflammatory activation to insulin resistance. CREBZF deficiency in macrophages, not in neutrophils, attenuates macrophage infiltration in adipose, proinflammatory activation, and hyperglycemia in diet-induced insulin-resistant mice. The coculture assays show that macrophage CREBZF deficiency improves insulin sensitivity in primary adipocytes and adipose tissue. Mechanistically, CREBZF competitively inhibits the binding of IκBα to p65, resulting in enhanced NF-κB activity. In addition, bromocriptine is identified as a small molecule inhibitor of CREBZF in macrophages, which suppresses the proinflammatory phenotype and improves metabolic dysfunction. Furthermore, CREBZF is highly expressed in ATM of obese humans and mice, which is positively correlated with proinflammatory genes and insulin resistance in humans. This study identifies a previously unknown role of CREBZF coupling ATM activation to systemic insulin resistance and type 2 diabetes.

Intergenerational relationship between parental famine exposure and offspring's obesity risks.

BACKGROUND: The intergenerational relationship between parental famine exposure and the obesity risks of offspring has not been well studied. METHODS: Using a cohort of 3654 respondents whose parents were born between 1950 and 1964 from the national data of the China Family Panel Studies, this study examines the associations between parental exposure to the 1959-1961 Chinese famine and offspring's body mass index (BMI). A cross-sectional difference-in-difference design was used to estimate the effects of parental famine exposures on offspring's BMI by exploiting temporal variations in the duration and period of famine across the parental birth cohorts between 1950 and 1964, and geographical variations in the famine severity at the province level. RESULTS: After adjusting individual characteristics and province-level fixed effects, we found that parental famine exposures in the preschool and school-age stages were associated with an increased BMI of offspring while there was not a significant association between prenatal famine exposure and offspring's BMI. The stratified analyses further show that the effects of parental famine exposure did not follow the same trajectory in subgroups. CONCLUSIONS: Famine experiences of parents were associated with increased BMI of offspring, suggesting an intergenerational impact of severe malnutrition on obesity risks.

Regulation of Hepatic Metabolism and Cell Growth by the ATF/CREB Family of Transcription Factors.

The liver is a major metabolic organ that regulates the whole-body metabolic homeostasis and controls hepatocyte proliferation and growth. The ATF/CREB family of transcription factors integrates nutritional and growth signals to the regulation of metabolism and cell growth in the liver, and deregulated ATF/CREB family signaling is implicated in the progression of type 2 diabetes, nonalcoholic fatty liver disease, and cancer. This article focuses on the roles of the ATF/CREB family in the regulation of glucose and lipid metabolism and cell growth and its importance in liver physiology. We also highlight how the disrupted ATF/CREB network contributes to human diseases and discuss the perspectives of therapeutically targeting ATF/CREB members in the clinic.

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.

The precursor of PI(3,4,5)P3 alleviates aging by activating daf-18(Pten) and independent of daf-16.

Aging is characterized by the loss of homeostasis and the general decline of physiological functions, accompanied by various degenerative diseases and increased rates of mortality. Aging targeting small molecule screens have been performed many times, however, few have focused on endogenous metabolic intermediates-metabolites. Here, using C. elegans lifespan assays, we conducted a worm metabolite screen and identified an eukaryotes conserved metabolite, myo-inositol (MI), to extend lifespan, increase mobility and reduce fat content. Genetic analysis of enzymes in MI metabolic pathway suggest that MI alleviates aging through its derivative PI(4,5)P2. MI and PI(4,5)P2 are precursors of PI(3,4,5)P3, which is negatively related to longevity. The longevity effect of MI is dependent on the tumor suppressor gene, daf-18 (homologous to mouse Pten), independent of its classical pathway downstream genes, akt or daf-16. Furthermore, we found MI effects on aging and lifespan act through mitophagy regulator PTEN induced kinase-1 (pink-1) and mitophagy. MI's anti-aging effect is also conserved in mouse, indicating a conserved mechanism in mammals.

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.

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.

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.

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.

Berberine attenuates hepatic steatosis and enhances energy expenditure in mice by inducing autophagy and fibroblast growth factor 21.

BACKGROUND AND PURPOSE: Berberine, a compound from rhizome coptidis, is traditionally used to treat gastrointestinal infections, such as bacterial diarrhoea. Recently, berberine was shown to have hypoglycaemic and hypolipidaemic effects. We investigated the mechanisms by which berberine regulates hepatic lipid metabolism and energy expenditure in mice. EXPERIMENTAL APPROACH: Liver-specific SIRT1 knockout mice and their wild-type littermates were fed a high-fat, high-sucrose (HFHS) diet and treated with berberine by i.p. injection for five weeks. Mouse primary hepatocytes and human HepG2 cells were treated with berberine and then subjected to immunoblotting analysis and Oil Red O staining. KEY RESULTS: Berberine attenuated hepatic steatosis and controlled energy balance in mice by inducing autophagy and FGF21. These beneficial effects of berberine on autophagy and hepatic steatosis were abolished by a deficiency of the nutrient sensor SIRT1 in the liver of HFHS diet-fed obese mice and in mouse primary hepatocytes. SIRT1 is essential for berberine to potentiate autophagy and inhibit lipid storage in mouse livers in response to fasting. Mechanistically, the berberine stimulates SIRT1 deacetylation activity and induces autophagy in an autophagy protein 5-dependent manner. Moreover, the administration of berberine was shown to promote hepatic gene expression and circulating levels of FGF21 and ketone bodies in mice in a SIRT1-dependent manner. CONCLUSIONS AND IMPLICATIONS: Berberine acts in the liver to regulate lipid utilization and maintain whole-body energy metabolism by mediating autophagy and FGF21 activation. Hence, it has therapeutic potential for treating metabolic defects under nutritional overload, such as fatty liver diseases, type 2 diabetes and obesity.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis.

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Hepatic ATF6 Increases Fatty Acid Oxidation to Attenuate Hepatic Steatosis in Mice Through Peroxisome Proliferator-Activated Receptor α.

The endoplasmic reticulum quality control protein activating transcription factor 6 (ATF6) has emerged as a novel metabolic regulator. Here, we show that adenovirus-mediated overexpression of the dominant-negative form of ATF6 (dnATF6) increases susceptibility to develop hepatic steatosis in diet-induced insulin-resistant mice and fasted mice. Overexpression of dnATF6 or small interfering RNA-mediated knockdown of ATF6 decreases the transcriptional activity of peroxisome proliferator-activated receptor α (PPARα)/retinoid X receptor complex, and inhibits oxygen consumption rates in hepatocytes, possibly through inhibition of the binding of PPARα to the promoter of its target gene. Intriguingly, ATF6 physically interacts with PPARα, enhances the transcriptional activity of PPARα, and triggers activation of PPARα downstream targets, such as CPT1α and MCAD, in hepatocytes. Furthermore, hepatic overexpression of the active form of ATF6 promotes hepatic fatty acid oxidation and protects against hepatic steatosis in diet-induced insulin-resistant mice. These data delineate the mechanism by which ATF6 controls the activity of PPARα and hepatic mitochondria fatty acid oxidation. Therefore, strategies to activate ATF6 could be used as an alternative avenue to improve liver function and treat hepatic steatosis in obesity.