Adipose lipolysis is important for ethanol to induce fatty liver in the National Institute on Alcohol Abuse and Alcoholism murine model of chronic and binge ethanol feeding.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) pathologies include steatosis, inflammation, and injury, which may progress to fibrosis, cirrhosis, and cancer. The liver receives ~60% of fatty acids from adipose tissue triglyceride hydrolysis, but the role of this lipolytic pathway in ALD development has not been directly examined in any genetic animal models with selective inactivation of adipose lipolysis. APPROACH AND RESULTS: Using adipose-specific comparative gene identification-58 (CGI-58) knockout (FAT-KO) mice, a model of impaired adipose lipolysis, we show that mice deficient in adipose lipolysis are almost completely protected against ethanol-induced hepatic steatosis and lipid peroxidation when subjected to the National Institute on Alcohol Abuse and Alcoholism chronic and binge ethanol feeding model. This is unlikely due to reduced lipid synthesis because this regimen of ethanol feeding down-regulated hepatic expression of lipogenic genes similarly in both genotypes. In the pair-fed group, FAT-KO relative to control mice displayed increased hepatocyte injury, neutrophil infiltration, and activation of the transcription factor signal transducer and activator of transcription 3 (STAT3) in the liver; and none of these were exacerbated by ethanol feeding. Activation of STAT3 is associated with a marked increase in hepatic leptin receptor mRNA expression and adipose inflammatory cell infiltration. CONCLUSIONS: Our findings establish a critical role of adipose lipolysis in driving hepatic steatosis and oxidative stress during ALD development.

Effects of UBE3A on Cell and Liver Metabolism through the Ubiquitination of PDHA1 and ACAT1.

Nonalcoholic fatty liver disease (NAFLD) is substantiated by the reprogramming of liver metabolic pathways that disrupts the homeostasis of lipid and glucose metabolism and thus promotes the progression of the disease. The metabolic pathways associated with NAFLD are regulated at different levels from gene transcription to various post-translational modifications including ubiquitination. Here, we used a novel orthogonal ubiquitin transfer platform to identify pyruvate dehydrogenase A1 (PDHA1) and acetyl-CoA acetyltransferase 1 (ACAT1), two important enzymes that regulate glycolysis and ketogenesis, as substrates of E3 ubiquitin ligase UBE3A/E6AP. We found that overexpression of UBE3A accelerated the degradation of PDHA1 and promoted glycolytic activities in HEK293 cells. Furthermore, a high-fat diet suppressed the expression of UBE3A in the mouse liver, which was associated with increased ACAT1 protein levels, while forced expression of UBE3A in the mouse liver resulted in decreased ACAT1 protein contents. As a result, the mice with forced expression of UBE3A in the liver exhibited enhanced accumulation of triglycerides, cholesterol, and ketone bodies. These results reveal the role of UBE3A in NAFLD development by inducing the degradation of ACAT1 in the liver and promoting lipid storage. Overall, our work uncovers an important mechanism underlying the regulation of glycolysis and lipid metabolism through UBE3A-mediated ubiquitination of PDHA1 and ACAT1 to regulate their stabilities and enzymatic activities in the cell.

Adipose Lipolysis Regulates Cardiac Glucose Uptake and Function in Mice under Cold Stress.

The heart primarily uses fatty acids as energy substrates. Adipose lipolysis is a major source of fatty acids, particularly under stress conditions. In this study, we showed that mice with selective inactivation of the lipolytic coactivator comparative gene identification-58 (CGI-58) in adipose tissue (FAT-KO mice), relative to their littermate controls, had lower circulating FA levels in the fed and fasted states due to impaired adipose lipolysis. They preferentially utilized carbohydrates as energy fuels and were more insulin sensitive and glucose tolerant. Under cold stress, FAT-KO versus control mice had >10-fold increases in glucose uptake in the hearts but no increases in other tissues examined. Plasma concentrations of atrial natriuretic peptide and cardiac mRNAs for atrial and brain-type natriuretic peptides, two sensitive markers of cardiac remodeling, were also elevated. After one week of cold exposure, FAT-KO mice showed reduced cardiac expression of several mitochondrial oxidative phosphorylation proteins. After one month of cold exposure, hearts of these animals showed depressed functions, reduced SERCA2 protein, and increased proteins for MHC-ß, collagen I proteins, Glut1, Glut4 and phospho-AMPK. Thus, CGI-58-dependent adipose lipolysis critically regulates cardiac metabolism and function, especially during cold adaptation. The adipose-heart axis may be targeted for the management of cardiac dysfunction.

Postnatal leptin surge is critical for the transient induction of the developmental beige adipocytes in mice.

Beige adipocytes have become a promising therapeutic target to combat obesity. Our senior author Dr. B. Xue previously discovered a transient but significant induction of beige adipocytes in mice during early postnatal development, which peaked at postnatal day (P) 20 and then disappeared thereafter. However, the physiological mechanism underlying the transient induction of the developmental beige cells remains mystery. Interestingly, there exists a postnatal surge of leptin in mice at P10 before the appearance of the developmental beige adipocytes. Given the neurotropic effect of leptin during neuronal development and its role in activating the sympathetic nervous system (SNS), we tested the hypothesis that postnatal leptin surge is required for the transient induction of developmental beige adipocytes through sympathetic innervation. Unlike wild-type (WT) mice that were able to acquire the developmentally induced beige adipocytes at P20, ob/ob mice had much less uncoupling protein 1 (UCP1)-positive multilocular cells in inguinal white adipose tissue at the same age. This was consistent with reduced expression of UCP1 mRNA and protein levels in white fat of ob/ob mice. In contrast, daily injection of ob/ob mice with leptin between P8 and P16, mimicking the postnatal leptin surge, largely rescued the ability of these mice to acquire the developmentally induced beige adipocytes at P20, which was associated with enhanced sympathetic nerve innervation assessed by whole mount adipose tissue immunostaining of tyrosine hydroxylase. Our data demonstrate that the postnatal leptin surge is essential for the developmentally induced beige adipocyte formation in mice, possibly through increasing sympathetic nerve innervation.

Histone Deacetylase 1 (HDAC1) Negatively Regulates Thermogenic Program in Brown Adipocytes via Coordinated Regulation of Histone H3 Lysine 27 (H3K27) Deacetylation and Methylation.

Inhibiting class I histone deacetylases (HDACs) increases energy expenditure, reduces adiposity, and improves insulin sensitivity in obese mice. However, the precise mechanism is poorly understood. Here, we demonstrate that HDAC1 is a negative regulator of the brown adipocyte thermogenic program. The Hdac1 level is lower in mouse brown fat (BAT) than white fat, is suppressed in mouse BAT during cold exposure or ß3-adrenergic stimulation, and is down-regulated during brown adipocyte differentiation. Remarkably, overexpressing Hdac1 profoundly blocks, whereas deleting Hdac1 significantly enhances, ß-adrenergic activation-induced BAT-specific gene expression in brown adipocytes. ß-Adrenergic activation in brown adipocytes results in a dissociation of HDAC1 from promoters of BAT-specific genes, including uncoupling protein 1 (Ucp1) and peroxisome proliferator-activated receptor γ co-activator 1α (Pgc1α), leading to increased acetylation of histone H3 lysine 27 (H3K27), an epigenetic mark of gene activation. This is followed by dissociation of the polycomb repressive complexes, including the H3K27 methyltransferase enhancer of zeste homologue (EZH2), suppressor of zeste 12 (SUZ12), and ring finger protein 2 (RNF2) from (and concomitant recruitment of H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) to) Ucp1 and Pgc1α promoters, leading to decreased H3K27 trimethylation, a histone transcriptional repression mark. Thus, HDAC1 negatively regulates the brown adipocyte thermogenic program, and inhibiting Hdac1 promotes BAT-specific gene expression through a coordinated control of increased acetylation and decreased methylation of H3K27, thereby switching the transcriptional repressive state to the active state at the promoters of Ucp1 and Pgc1α. Targeting HDAC1 may be beneficial in prevention and treatment of obesity by enhancing BAT thermogenesis.

Bidirectional crosstalk between the sensory and sympathetic motor systems innervating brown and white adipose tissue in male Siberian hamsters.

The brain networks connected to the sympathetic motor and sensory innervations of brown (BAT) and white (WAT) adipose tissues were originally described using two transneuronally transported viruses: the retrogradely transported pseudorabies virus (PRV), and the anterogradely transported H129 strain of herpes simplex virus-1 (HSV-1 H129). Further complexity was added to this network organization when combined injections of PRV and HSV-1 H129 into either BAT or WAT of the same animal generated sets of coinfected neurons in the brain, spinal cord, and sympathetic and dorsal root ganglia. These neurons are well positioned to act as sensorimotor links in the feedback circuits that control each fat pad. We have now determined the extent of sensorimotor crosstalk between interscapular BAT (IBAT) and inguinal WAT (IWAT). PRV152 and HSV-1 H129 were each injected into IBAT or IWAT of the same animal: H129 into IBAT and PRV152 into IWAT. The reverse configuration was applied in a different set of animals. We found single-labeled neurons together with H129+PRV152 coinfected neurons in multiple brain sites, with lesser numbers in the sympathetic and dorsal root ganglia that innervate IBAT and IWAT. We propose that these coinfected neurons mediate sensory-sympathetic motor crosstalk between IBAT and IWAT. Comparing the relative numbers of coinfected neurons between the two injection configurations showed a bias toward IBAT-sensory and IWAT-sympathetic motor feedback loops. These coinfected neurons provide a neuroanatomical framework for functional interactions between IBAT thermogenesis and IWAT lipolysis that occurs with cold exposure, food restriction/deprivation, exercise, and more generally with alterations in adiposity.

Separate and shared sympathetic outflow to white and brown fat coordinately regulates thermoregulation and beige adipocyte recruitment.

White adipose tissue (WAT) and brown adipose tissue (BAT) are innervated and regulated by the sympathetic nervous system (SNS). It is not clear, however, whether there are shared or separate central SNS outflows to WAT and BAT that regulate their function. We injected two isogenic strains of pseudorabies virus, a retrograde transneuronal viral tract tracer, with unique fluorescent reporters into interscapular BAT (IBAT) and inguinal WAT (IWAT) of the same Siberian hamsters to define SNS pathways to both. To test the functional importance of SNS coordinated control of BAT and WAT, we exposed hamsters with denervated SNS nerves to IBAT to 4°C for 16-24 h and measured core and fat temperatures and norepinephrine turnover (NETO) and uncoupling protein 1 (UCP1) expression in fat tissues. Overall, there were more SNS neurons innervating IBAT than IWAT across the neuroaxis. However, there was a greater percentage of singly labeled IWAT neurons in midbrain reticular nuclei than singly labeled IBAT neurons. The hindbrain had ~30-40% of doubly labeled neurons while the forebrain had ~25% suggesting shared SNS circuitry to BAT and WAT across the brain. The raphe nucleus, a key region in thermoregulation, had ~40% doubly labeled neurons. Hamsters with IBAT SNS denervation maintained core body temperature during acute cold challenge and had increased beige adipocyte formation in IWAT. They also had increased IWAT NETO, temperature, and UCP1 expression compared with intact hamsters. These data provide strong neuroanatomical and functional evidence of WAT and BAT SNS cross talk for thermoregulation and beige adipocyte formation.

The Histone Demethylase UTX Promotes Brown Adipocyte Thermogenic Program Via Coordinated Regulation of H3K27 Demethylation and Acetylation.

Brown adipocytes function to dissipate energy as heat through adaptive thermogenesis. Understanding the molecular mechanisms underlying the brown fat thermogenic program may provide insights for the development of therapeutic approaches in the treatment of obesity. Most studies investigating the mechanisms underlying brown fat development focus on genetic mechanisms; little is known about the epigenetic mechanisms in this process. We have discovered that ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), a histone demethylase for di- or tri-methylated histone 3 lysine 27 (H3K27me2/3), plays a potential role in regulating brown adipocyte thermogenic program. We found that UTX is up-regulated during brown adipocyte differentiation and by cold exposure in both brown adipose tissue (BAT) and white adipose tissue (WAT) of mice, suggesting a potential role in thermogenesis. Inactivation of UTX down-regulates brown fat specific gene expression, while overexpression of UTX does the opposite. Notably, activation of ß adrenergic signaling recruits UTX to the UCP1 and PGC1α promoters, leading to decreased H3K27me3, a histone transcriptional repressive mark. UTX demethylates H3K27me3 and subsequently interacts with the histone acetyltransferase (HAT) protein CBP, resulting in increased H3K27 acetylation (H3K27ac), a histone transcriptional active mark. UTX positively regulate brown adipocyte thermogenic program through coordinated control of demethylating H3K27me3 and acetylating H3K27, switching the transcriptional repressive state to the transcriptional active state at the promoters of UCP1 and PGC1α. We conclude that UTX may play a potential role in regulation of brown adipocyte gene expression and may mediate ß adrenergic activation of brown fat function.

Macrophage CGI-58 deficiency promotes IL-1ß transcription by activating the SOCS3-FOXO1 pathway.

Over-nutrition induces low-grade inflammation that dampens insulin sensitivity, but the underlying molecular mediators are not fully understood. Comparative gene identification-58 (CGI-58) is an intracellular lipolytic activator. In the present study, we show that in mouse visceral fat-derived macrophages or human peripheral blood monocytes, CGI-58 negatively and interleukin (IL)-1ß positively correlate with obesity. Saturated non-esterified fatty acid (NEFA) suppresses CGI-58 expression in macrophages and this suppression activates FOXO1 (forkhead box-containing protein O subfamily-1) through inhibition of FOXO1 phosphorylation. Activated FOXO1 binds to an insulin-responsive element in IL-1ß promoter region to potentiate IL-1ß transcription. Gain- and loss-of-function studies demonstrate that NEFA-induced CGI-58 suppression activates FOXO1 to augment IL-1ß transcription by dampening insulin signalling through induction of SOCS3 (suppressor of cytokine signalling 3) expression. CGI-58 deficiency-induced SOCS3 expression is NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome-dependent. Our data thus identified a vicious cycle (IL-1ß-SOCS3-FOXO1-IL-1ß) that amplifies IL-1ß secretion and is initiated by CGI-58 deficiency-induced activation of the NLRP3 inflammasome in macrophages. We further show that blocking this cycle with a FOXO1 inhibitor, an antioxidant that inhibits FOXO1 or IL-1 receptor antagonist alleviates chronic inflammation and insulin resistance in high-fat diet (HFD)-fed mice. Collectively, our data suggest that obesity-associated factors such as NEFA and lipopolysaccharide (LPS) probably adopt this vicious cycle to promote inflammation and insulin resistance.

Protein kinase C-δ mediates sepsis-induced activation of complement 5a and urokinase-type plasminogen activator signaling in macrophages.

OBJECTIVE AND DESIGN: Activations of the complement C5a (C5a) and the urokinase-type plasminogen activator (uPA) are commonly seen together during sepsis. However, the mechanism linking these two important pathways remains elusive. MATERIAL, METHODS AND TREATMENT: We used the C57BL/6 J mice model of sepsis induced by cecal ligation puncture (CLP) procedure, injected anti-C5aR or rottlerin through the tail vein to neutralize C5aR or PKC-δ, and then isolated peritoneal macrophages. Total RNA was isolated from the cells and analyzed by quantitative PCR. RESULTS: Our study revealed that neutralizing C5aR markedly inhibited sepsis-induced uPA receptor (uPAR) expression and its downstream signaling in macrophage. Similarly, neutralizing uPAR suppressed sepsis activation of C5a signaling. Importantly, inhibition of PKC-δ largely blocked sepsis-induced expression of C5aR and uPAR. CONCLUSIONS: Our study demonstrates a crosstalk between the complement C5a signaling and the fibrinolytic uPA pathways, which may depend on each other to maintain their expression and signaling, and reveals a central role of PKC-δ in mediating sepsis-induced activation of these pathways.

Disturbed flow impairs MerTK-mediated efferocytosis in aortic endothelial cells during atherosclerosis.

Background: MER proto-oncogene tyrosine kinase (MerTK) is a key receptor for efferocytosis, a process for the clearance of apoptotic cells. MerTK is mainly expressed in macrophages and immature dendritic cells. There are very limited reports focused on MerTK biology in aortic endothelial cells (ECs). It remains unclear for the role of blood flow patterns in regulating MerTK-mediated efferocytosis in aortic ECs. This study was designed to investigate whether endothelial MerTK and EC efferocytosis respond to blood flow patterns during atherosclerosis. Methods: Big data analytics, RNA-seq and proteomics combined with our in vitro and in vivo studies were applied to reveal the potential molecular mechanisms. Partial carotid artery ligation combined with AAV-PCSK9 and high fat diet were used to set up acute atherosclerosis in 4 weeks. Results: Our data showed that MerTK is sensitive to blood flow patterns and is inhibited by disturbed flow and oscillatory shear stress in primary human aortic ECs (HAECs). The RNA-seq data in HAECs incubated with apoptotic cells showed that d-flow promotes pro-inflammatory pathway and senescence pathway. Our in vivo data of proteomics and immunostaining showed that, compared with WT group, MerTK-/- aggravates atherosclerosis in d-flow areas through upregulation of endothelial dysfunction markers (e.g. IL-1ß, NF-κB, TLR4, MAPK signaling, vWF, VCAM-1 and p22phox) and mitochondrial dysfunction. Interestingly, MerTK-/-induces obvious abnormal endothelial thickening accompanied with decreased endothelial efferocytosis, promoting the development of atherosclerosis. Conclusions: Our data suggests that blood flow patterns play an important role in regulating MerTK-mediated efferocytosis in aortic ECs, revealing a new promising therapeutic strategy with EC efferocytosis restoration to against atherosclerosis.

Big data analytics for MerTK genomics reveals its double-edged sword functions in human diseases.

RATIONALE: MER proto-oncogene tyrosine kinase (MerTK) is a key receptor for the clearance of apoptotic cells (efferocytosis) and plays important roles in redox-related human diseases. We will explore MerTK biology in human cells, tissues, and diseases based on big data analytics. METHODS: The human RNA-seq and scRNA-seq data about 42,700 samples were from NCBI Gene Expression Omnibus and analyzed by QIAGEN Ingenuity Pathway Analysis (IPA) with about 170,000 crossover analysis. MerTK expression was quantified as Log2 (FPKM + 0.1). RESULTS: We found that, in human cells, MerTK is highly expressed in macrophages, monocytes, progenitor cells, alpha-beta T cells, plasma B cells, myeloid cells, and endothelial cells (ECs). In human tissues, MerTK has higher expression in plaque, blood vessels, heart, liver, sensory system, artificial tissue, bone, adrenal gland, central nervous system (CNS), and connective tissue. Compared to normal conditions, MerTK expression in related tissues is altered in many human diseases, including cardiovascular diseases, cancer, and brain disorders. Interestingly, MerTK expression also shows sex differences in many tissues, indicating that MerTK may have different impact on male and female. Finally, based on our proteomics from primary human aortic ECs, we validated the functions of MerTK in several human diseases, such as cancer, aging, kidney failure and heart failure. CONCLUSIONS: Our big data analytics suggest that MerTK may be a promising therapeutic target, but how it should be modulated depends on the disease types and sex differences. For example, MerTK inhibition emerges as a new strategy for cancer therapy due to it counteracts effect on anti-tumor immunity, while MerTK restoration represents a promising treatment for atherosclerosis and myocardial infarction as MerTK is cleaved in these disease conditions.

The histone methyltransferase SUV420H2 regulates brown and beige adipocyte thermogenesis.

Activation of brown adipose tissue (BAT) thermogenesis increases energy expenditure and alleviates obesity. Here we discover that histone methyltransferase suppressor of variegation 4-20 homolog 2 (Suv420h2) expression parallels that of Ucp1 in brown and beige adipocytes and that Suv420h2 knockdown significantly reduces - whereas Suv420h2 overexpression significantly increases - Ucp1 levels in brown adipocytes. Suv420h2 knockout (H2KO) mice exhibit impaired cold-induced thermogenesis and are prone to diet-induced obesity. In contrast, mice with specific overexpression of Suv420h2 in adipocytes display enhanced cold-induced thermogenesis and are resistant to diet-induced obesity. Further study shows that Suv420h2 catalyzes H4K20 trimethylation at eukaryotic translation initiation factor 4E-binding protein 1 (4e-bp1) promoter, leading to downregulated expression of 4e-bp1, a negative regulator of the translation initiation complex. This in turn upregulates PGC1α protein levels, and this upregulation is associated with increased expression of thermogenic program. We conclude that Suv420h2 is a key regulator of brown/beige adipocyte development and thermogenesis.

Deficiency of liver Comparative Gene Identification-58 causes steatohepatitis and fibrosis in mice.

Triglyceride (TG) accumulation in hepatocytes (hepatic steatosis) preludes the development of advanced nonalcoholic fatty liver diseases (NAFLDs) such as steatohepatitis, fibrosis, and cirrhosis. Mutations in human Comparative Gene Identification-58 (CGI-58) cause cytosolic TG-rich lipid droplets to accumulate in almost all cell types including hepatocytes. However, it is unclear if CGI-58 mutation causes hepatic steatosis locally or via altering lipid metabolism in other tissues. To directly address this question, we created liver-specific CGI-58 knockout (LivKO) mice. LivKO mice on standard chow diet displayed microvesicular and macrovesicular panlobular steatosis, and progressed to advanced NAFLD stages over time, including lobular inflammation and centrilobular fibrosis. Compared with CGI-58 floxed control littermates, LivKO mice showed 8-fold and 52-fold increases in hepatic TG content, which was associated with 40% and 58% decreases in hepatic TG hydrolase activity at 16 and 42 weeks, respectively. Hepatic cholesterol also increased significantly in LivKO mice. At 42 weeks, LivKO mice showed increased hepatic oxidative stress, plasma aminotransferases, and hepatic mRNAs for genes involved in fibrosis and inflammation, such as α-smooth muscle actin, collagen type 1 α1, tumor necrosis factor α, and interleukin-1ß. In conclusion, CGI-58 deficiency in the liver directly causes not only hepatic steatosis but also steatohepatitis and fibrosis.

Neuronal PTP1B regulates body weight, adiposity and leptin action.

Obesity is a major health problem and a risk factor for type 2 diabetes. Leptin, an adipocyte-secreted hormone, acts on the hypothalamus to inhibit food intake and increase energy expenditure. Most obese individuals develop hyperleptinemia and leptin resistance, limiting the therapeutic efficacy of exogenously administered leptin. Mice lacking the tyrosine phosphatase PTP1B are protected from diet-induced obesity and are hypersensitive to leptin, but the site and mechanism for these effects remain controversial. We generated tissue-specific PTP1B knockout (Ptpn1(-/-)) mice. Neuronal Ptpn1(-/-) mice have reduced weight and adiposity, and increased activity and energy expenditure. In contrast, adipose PTP1B deficiency increases body weight, whereas PTP1B deletion in muscle or liver does not affect weight. Neuronal Ptpn1(-/-) mice are hypersensitive to leptin, despite paradoxically elevated leptin levels, and show improved glucose homeostasis. Thus, PTP1B regulates body mass and adiposity primarily through actions in the brain. Furthermore, neuronal PTP1B regulates adipocyte leptin production and probably is essential for the development of leptin resistance.

Chronic Exposure to Low-Molecular-Weight Polycyclic Aromatic Hydrocarbons Promotes Lipid Accumulation and Metabolic Inflammation.

2-naphthol is a low-molecular-weight (LMW) polycyclic aromatic hydrocarbon (PAH) and air pollutant associated with childhood obesity. There has been a recent emergence of studies on the consequences of PAHs on human health. Current epidemiological reports suggest LMW-PAHs may contribute to obesity incidences in children, yet most studies focus on high-molecular-weight PAHs. This study explores 2-naphthol's impact on obesity and obesity-associated metabolic disorders. To investigate 2-naphthol's effect on lipid metabolism and inflammation, we employed 3T3-L1 and BAT1 cell lines to model white and brown adipocytes, respectively, alongside a murine macrophage cell line (RAW264.7). We found that 2-naphthol increased the expression of key adipogenic and lipogenic genes while decreasing lipolytic gene expression in chronically treated 3T3-L1 and BAT1 adipocytes. In addition, chronic 2-naphthol treatment also suppressed adrenergic-stimulated thermogenic gene expression in BAT1 brown adipocytes. In consistence, an increase in lipid accumulation was demonstrated in BODIPY and Oil Red O-stained adipocytes. Additionally, 3T3-L1 adipocytes and RAW264.7 macrophages chronically exposed to 2-naphthol showed upregulated mRNA expression of major inflammatory cytokines (e.g., tumor necrosis factor α (Tnfα), interleukin-1ß (Il-1ß), and Il-6). In summary, chronic exposure to 2-naphthol stimulates lipid accumulation in adipocytes and inflammation in adipocytes and macrophages. These findings support previous research that demonstrates 2-naphthol has obesogenic potential.

Single ethanol binge causes severe liver injury in mice fed Western diet.

BACKGROUND AND AIMS: Alcohol-associated liver disease (ALD) and NAFLD often coexist in Western societies that consume energy-rich and cholesterol-containing Western diets. Increased rates of ALD mortality in young people in these societies are likely attributable to binge drinking. It is largely unknown how alcohol binge causes liver damage in the setting of Western diets. APPROACH AND RESULTS: In this study, we showed that a single ethanol binge (5 g/kg body weight) induced severe liver injury as shown by marked increases in serum activities of the 2 aminotransferases AST and ALT in C57BL/6J mice that have been fed a Western diet for 3 weeks. The Western diet plus binge ethanol-fed mice also displayed severe lipid droplet deposition and high contents of triglycerides and cholesterol in the liver, which were associated with increased lipogenic and reduced fatty acid oxidative gene expression. These animals had the highest Cxcl1 mRNA expression and myeloperoxidase (MPO)-positive neutrophils in the liver. Their hepatic ROS and lipid peroxidation were the highest, but their hepatic levels of mitochondrial oxidative phosphorylation proteins remained largely unaltered. Hepatic levels of several ER stress markers, including mRNAs for CHOP, ERO1A, ERO1B, BIM, and BIP, as well as Xbp1 splicing and proteins for BIP/GRP78 and IRE-α were also the highest in these animals. Interestingly, Western diet feeding for 3 weeks or ethanol binge dramatically increased hepatic caspase 3 cleavage, and the combination of the 2 did not further increase it. Thus, we successfully established a murine model of acute liver injury by mimicking human diets and binge drinking. CONCLUSIONS: This simple Western diet plus single ethanol binge model recapitulates major hepatic phenotypes of ALD, including steatosis and steatohepatitis characterized by neutrophil infiltration, oxidative stress, and ER stress.

Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

While extensive investigations have been devoted to the study of genetic pathways related to fatty liver diseases, much less is known about epigenetic mechanisms underlying these disorders. DNA methylation is an epigenetic link between environmental factors (e.g., diets) and complex diseases (e.g., non-alcoholic fatty liver disease). Here, it is aimed to study the role of DNA methylation in the regulation of hepatic lipid metabolism. A dynamic change in the DNA methylome in the liver of high-fat diet (HFD)-fed mice is discovered, including a marked increase in DNA methylation at the promoter of Beta-klotho (Klb), a co-receptor for the biological functions of fibroblast growth factor (FGF)15/19 and FGF21. DNA methyltransferases (DNMT) 1 and 3A mediate HFD-induced methylation at the Klb promoter. Notably, HFD enhances DNMT1 protein stability via a ubiquitination-mediated mechanism. Liver-specific deletion of Dnmt1 or 3a increases Klb expression and ameliorates HFD-induced hepatic steatosis. Single-nucleus RNA sequencing analysis reveals pathways involved in fatty acid oxidation in Dnmt1-deficient hepatocytes. Targeted demethylation at the Klb promoter increases Klb expression and fatty acid oxidation, resulting in decreased hepatic lipid accumulation. Up-regulation of methyltransferases by HFD may induce hypermethylation of the Klb promoter and subsequent down-regulation of Klb expression, resulting in the development of hepatic steatosis.

Crosstalk between Gut Sensory Ghrelin Signaling and Adipose Tissue Sympathetic Outflow Regulates Metabolic Homeostasis.

The stomach-derived orexigenic hormone ghrelin is a key regulator of energy homeostasis and metabolism in humans. The ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR), is widely expressed in the brain and gastrointestinal vagal sensory neurons, and neuronal GHSR knockout results in a profoundly beneficial metabolic profile and protects against diet-induced obesity (DIO) and insulin resistance. Here we show that in addition to the well characterized vagal GHSR, GHSR is robustly expressed in gastrointestinal sensory neurons emanating from spinal dorsal root ganglia. Remarkably, sensory neuron GHSR deletion attenuates DIO through increased energy expenditure and sympathetic outflow to adipose tissue independent of food intake. In addition, neuronal viral tract tracing reveals prominent crosstalk between gut non-vagal sensory afferents and adipose sympathetic outflow. Hence, these findings demonstrate a novel gut sensory ghrelin signaling pathway critical for maintaining energy homeostasis.

Neuronal GHS-R Differentially Modulates Feeding Patterns under Normal and Obesogenic Conditions.

The orexigenic hormone ghrelin increases food intake and promotes obesity through its receptor, growth hormone secretagogue receptor (GHS-R). We previously reported two neuron-specific GHS-R knockout mouse lines, namely pan-neuronal deletion by Syn1-cre and hypothalamic deletion by AgRP-cre, exhibiting differential diet-dependent effects on body weight. GHS-R deficiency in neurons elicited less pronounced metabolic effects under regular diet (RD) than high fat diet (HFD). While there was no difference in total food intake of HFD in either mouse line, Syn1-cre; Ghsrf/f mice showed much greater anti-obesity effect than that of AgRP-cre; Ghsrf/f mice. Meal feeding pattern is known to have a major impact on energy homeostasis and obesity development. Here, we investigated the feeding behaviors of these two neuron-specific GHS-R knockout mice under RD and HFD feeding, by assessing meal number, meal size, meal duration, and feeding frequency. Under the normal diet, RD-fed Syn1-cre; Ghsrf/f mice showed a decreased meal size in dark phase, while RD-fed AgRP-cre; Ghsrf/f mice showed an increased meal duration in dark phase. Under the obesogenic diet, HFD-fed Syn1-cre; Ghsrf/f mice displayed reduced meal numbers in light phase and increased feeding in both light and dark phases, whereas HFD-fed AgRP-cre; Ghsrf/f mice showed a decreased meal duration in the light phase only. Consistently, the expression of neuropeptides (Neuropeptide Y and Orexin) was increased in the hypothalamus of RD-fed Syn1-cre; Ghsrf/f mice, whereas the expression of cannabinoid receptor type 1 (CB1) was increased in the hypothalamus of HFD fed Syn1-cre; Ghsrf/f mice. Overall, feeding pattern changes were more pronounced in Syn1-cre; Ghsrf/f mice than that in AgRP-cre; Ghsrf/f mice, and HFD elicited greater alteration than RD. While AgRP-cre; Ghsrf/f mice consumed HFD meals faster during the day (showing shorter meal duration), Syn1-cre; Ghsrf/f mice ate few HFD meals during the light phase and ate slowly throughout the day (showing longer meal duration in both phases). Our findings reveal that neuronal GHS-R regulates energy homeostasis by altering feeding patterns, and differentially modulates feeding patterns in a site- and diet-dependent manner. The distinctive data in these two mouse lines also suggest that eating slowly during the optimal feeding period (dark phase for mice) may be beneficial in combating obesity.