Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis.

Cell-cell communication via ligand-receptor signaling is a fundamental feature of complex organs. Despite this, the global landscape of intercellular signaling in mammalian liver has not been elucidated. Here we perform single-cell RNA sequencing on non-parenchymal cells isolated from healthy and NASH mouse livers. Secretome gene analysis revealed a highly connected network of intrahepatic signaling and disruption of vascular signaling in NASH. We uncovered the emergence of NASH-associated macrophages (NAMs), which are marked by high expression of triggering receptors expressed on myeloid cells 2 (Trem2), as a feature of mouse and human NASH that is linked to disease severity and highly responsive to pharmacological and dietary interventions. Finally, hepatic stellate cells (HSCs) serve as a hub of intrahepatic signaling via HSC-derived stellakines and their responsiveness to vasoactive hormones. These results provide unprecedented insights into the landscape of intercellular crosstalk and reprogramming of liver cells in health and disease.

Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

Myeloid-specific ablation of Basp1 ameliorates diet-induced NASH in mice by attenuating pro-inflammatory signaling.

BACKGROUND AND AIMS: NASH represents a severe stage of fatty liver disease characterized by hepatocyte injury, inflammation, and liver fibrosis. Myeloid-derived innate immune cells, such as macrophages and dendritic cells, play an important role in host defense and disease pathogenesis. Despite this, the nature of transcriptomic reprogramming of myeloid cells in NASH liver and its contribution to disease progression remain incompletely defined. APPROACH AND RESULTS: In this study, we performed bulk and single-cell RNA sequencing (sc-RNA seq) analysis to delineate the landscape of macrophage and dendritic cell transcriptomes in healthy and NASH livers. Our analysis uncovered cell type-specific patterns of transcriptomic reprogramming on diet-induced NASH. We identified brain-abundant membrane-attached signal protein 1 (Basp1) as a myeloid-enriched gene that is markedly induced in mouse and human NASH liver. Myeloid-specific inactivation of Basp1 attenuates the severity of diet-induced NASH pathologies, as shown by reduced hepatocyte injury and liver fibrosis in mice. Mechanistically, cultured macrophages lacking Basp1 exhibited a diminished response to pro-inflammatory stimuli, impaired NLRP3 inflammasome activation, and reduced cytokine secretion. CONCLUSIONS: Together, these findings uncover Basp1 as a critical regulator of myeloid inflammatory signaling that underlies NASH pathogenesis.

Glucose Sensing by Skeletal Myocytes Couples Nutrient Signaling to Systemic Homeostasis.

Skeletal muscle is a major site of postprandial glucose disposal. Inadequate insulin action in skeletal myocytes contributes to hyperglycemia in diabetes. Although glucose is known to stimulate insulin secretion by ß cells, whether it directly engages nutrient signaling pathways in skeletal muscle to maintain systemic glucose homeostasis remains largely unexplored. Here we identified the Baf60c-Deptor-AKT pathway as a target of muscle glucose sensing that augments insulin action in skeletal myocytes. Genetic activation of this pathway improved postprandial glucose disposal in mice, whereas its muscle-specific ablation impaired insulin action and led to postprandial glucose intolerance. Mechanistically, glucose triggers KATP channel-dependent calcium signaling, which promotes HDAC5 phosphorylation and nuclear exclusion, leading to Baf60c induction and insulin-independent AKT activation. This pathway is engaged by the anti-diabetic sulfonylurea drugs to exert their full glucose-lowering effects. These findings uncover an unexpected mechanism of glucose sensing in skeletal myocytes that contributes to homeostasis and therapeutic action.

Intrahepatic paracrine signaling by cardiotrophin-like cytokine factor 1 ameliorates diet-induced NASH in mice.

BACKGROUND AND AIMS: The mammalian liver harbors heterogeneous cell types that communicate via local paracrine signaling. Recent studies have delineated the transcriptomic landscape of the liver in NASH that provides insights into liver cell heterogeneity, intercellular crosstalk, and disease-associated reprogramming. However, the nature of intrahepatic signaling and its role in NASH progression remain obscure. APPROACH AND RESULTS: Here, we performed transcriptomic analyses and identified cardiotrophin-like cytokine factor 1 (CLCF1), a member of the IL-6 family cytokines, as a cholangiocyte-derived paracrine factor that was elevated in the liver from diet-induced NASH mice and patients with NASH. Adenovirus-associated virus-mediated overexpression of CLCF1 in the liver ameliorated NASH pathologies in two diet-induced NASH models in mice, illustrating that CLCF1 induction may serve an adaptive and protective role during NASH pathogenesis. Unexpectedly, messenger RNA and protein levels of leukemia inhibitory factor receptor (LIFR), a subunit of the receptor complex for CLCF1, were markedly downregulated in NASH liver. Hepatocyte-specific inactivation of LIFR accelerated NASH progression in mice, supporting an important role of intrahepatic cytokine signaling in maintaining tissue homeostasis under metabolic stress conditions. CONCLUSIONS: Together, this study sheds light on the molecular nature of intrahepatic paracrine signaling during NASH pathogenesis and uncovers potential targets for therapeutic intervention.

Hepatic mitochondrial NAD + transporter SLC25A47 activates AMPKα mediating lipid metabolism and tumorigenesis.

BACKGROUND AIMS: SLC25A47 was initially identified as a mitochondrial HCC-downregulated carrier protein, but its physiological functions and transport substrates are unknown. We aimed to investigate the physiological role of SLC25A47 in hepatic metabolism. APPROACH RESULTS: In the treatment of hepatocytes with metformin, we found that metformin can transcriptionally activate the expression of Slc25a47 , which is required for AMP-activated protein kinase α (AMPKα) phosphorylation. Slc25a47 -deficient mice had increased hepatic lipid content, triglycerides, and cholesterol levels, and we found that Slc25a47 deficiency suppressed AMPKα phosphorylation and led to an increased accumulation of nuclear SREBPs, with elevated fatty acid and cholesterol biosynthetic activities. Conversely, when Slc25a47 was overexpressed in mouse liver, AMPKα was activated and resulted in the inhibition of lipogenesis. Moreover, using a diethylnitrosamine-induced mouse HCC model, we found that the deletion of Slc25a47 promoted HCC tumorigenesis and development through the activated mammalian target of rapamycin cascade. Employing homology modeling of SLC25A47 and virtual screening of the human metabolome database, we demonstrated that NAD + was an endogenous substrate for SLC25A47, and the activity of NAD + -dependent sirtuin 3 declined in Slc25a47 -deficient mice, followed by inactivation of AMPKα. CONCLUSIONS: Our findings reveal that SLC25A47, a hepatocyte-specific mitochondrial NAD + transporter, is one of the pharmacological targets of metformin and regulates lipid homeostasis through AMPKα, and may serve as a potential drug target for treating NAFLD and HCC.

Reprogramming of Hepatic Metabolism and Microenvironment in Nonalcoholic Steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD), a spectrum of metabolic liver disease associated with obesity, ranges from relatively benign hepatic steatosis to nonalcoholic steatohepatitis (NASH). The latter is characterized by persistent liver injury, inflammation, and liver fibrosis, which collectively increase the risk for end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. Recent work has shed new light on the pathophysiology of NAFLD/NASH, particularly the role of genetic, epigenetic, and dietary factors and metabolic dysfunctions in other tissues in driving excess hepatic fat accumulation and liver injury. In parallel, single-cell RNA sequencing studies have revealed unprecedented details of the molecular nature of liver cell heterogeneity, intrahepatic cross talk, and disease-associated reprogramming of the liver immune and stromal vascular microenvironment. This review covers the recent advances in these areas, the emerging concepts of NASH pathogenesis, and potential new therapeutic opportunities.

A long noncoding RNA transcriptional regulatory circuit drives thermogenic adipocyte differentiation.

Brown and beige/brite fats generate heat via uncoupled respiration to defend against cold. The total mass and activity of thermogenic adipose tissues are also tightly linked to systemic energy and nutrient homeostasis. Despite originating from distinct progenitors, brown and beige adipocytes acquire remarkably similar molecular and metabolic characteristics during differentiation through the action of a network of transcription factors and cofactors. How this regulatory network interfaces with long noncoding RNAs (lncRNAs), an emerging class of developmental regulators, remains largely unexplored. Here, we globally profiled lncRNA gene expression during thermogenic adipocyte formation and identified Brown fat lncRNA 1 (Blnc1) as a nuclear lncRNA that promotes brown and beige adipocyte differentiation and function. Blnc1 forms a ribonucleoprotein complex with transcription factor EBF2 to stimulate the thermogenic gene program. Further, Blnc1 itself is a target of EBF2, thereby forming a feedforward regulatory loop to drive adipogenesis toward thermogenic phenotype.

hnRNPU/TrkB Defines a Chromatin Accessibility Checkpoint for Liver Injury and Nonalcoholic Steatohepatitis Pathogenesis.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is a progressive liver disease that is characterized by liver injury, inflammation, and fibrosis. NASH pathogenesis is linked to reprogramming of chromatin landscape in the liver that predisposes hepatocytes to stress-induced tissue injury. However, the molecular nature of the putative checkpoint that maintains chromatin architecture and preserves hepatocyte health remains elusive. APPROACH AND RESULTS: Here we show that heterogeneous nuclear ribonucleoprotein U (hnRNPU), a nuclear matrix protein that governs chromatin architecture and gene transcription, is a critical factor that couples chromatin disruption to NASH pathogenesis. RNA-seq and chromatin immunoprecipitation-seq studies revealed an extensive overlap between hnRNPU occupancy and altered gene expression during NASH. Hepatocyte-specific inactivation of hnRNPU disrupted liver chromatin accessibility, activated molecular signature of NASH, and sensitized mice to diet-induced NASH pathogenesis. Mechanistically, hnRNPU deficiency stimulated the expression of a truncated isoform of TrkB (TRKB-T1) that promotes inflammatory signaling in hepatocytes and stress-induced cell death. Brain-derived neurotrophic factor treatment reduced membrane TRKB-T1 protein and protected mice from diet-induced NASH. CONCLUSIONS: These findings illustrate a mechanism through which disruptions of chromatin architecture drive the emergence of disease-specific signaling patterns that promote liver injury and exacerbate NASH pathogenesis.

BAF60a Deficiency in Vascular Smooth Muscle Cells Prevents Abdominal Aortic Aneurysm by Reducing Inflammation and Extracellular Matrix Degradation.

OBJECTIVE: Currently, there are no approved drugs for abdominal aortic aneurysm (AAA) treatment, likely due to limited understanding of the primary molecular mechanisms underlying AAA development and progression. BAF60a-a unique subunit of the SWI/SNF (switch/sucrose nonfermentable) chromatin remodeling complex-is a novel regulator of metabolic homeostasis, yet little is known about its function in the vasculature and pathogenesis of AAA. In this study, we sought to investigate the role and underlying mechanisms of vascular smooth muscle cell (VSMC)-specific BAF60a in AAA formation. Approach and Results: BAF60a is upregulated in human and experimental murine AAA lesions. In vivo studies revealed that VSMC-specific knockout of BAF60a protected mice from both Ang II (angiotensin II)-induced and elastase-induced AAA formation with significant suppression of vascular inflammation, monocyte infiltration, and elastin fragmentation. Through RNA sequencing and pathway analysis, we found that the expression of inflammatory response genes in cultured human aortic smooth muscle cells was significantly downregulated by small interfering RNA-mediated BAF60a knockdown while upregulated upon adenovirus-mediated BAF60a overexpression. BAF60a regulates VSMC inflammation by recruiting BRG1 (Brahma-related gene-1)-a catalytic subunit of the SWI/SNF complex-to the promoter region of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) target genes. Furthermore, loss of BAF60a in VSMCs prevented the upregulation of the proteolytic enzyme cysteine protease CTSS (cathepsin S), thus ameliorating ECM (extracellular matrix) degradation within the vascular wall in AAA. CONCLUSIONS: Our study demonstrated that BAF60a is required to recruit the SWI/SNF complex to facilitate the epigenetic regulation of VSMC inflammation, which may serve as a potential therapeutic target in preventing and treating AAA.

Inhibition of AMPK catabolic action by GSK3.

AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK ß regulatory subunit and phosphorylates the AMPK α catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the α subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK.

KDM4B protects against obesity and metabolic dysfunction.

Although significant progress has been made in understanding epigenetic regulation of in vitro adipogenesis, the physiological functions of epigenetic regulators in metabolism and their roles in obesity remain largely elusive. Here, we report that KDM4B (lysine demethylase 4B) in adipose tissues plays a critical role in energy balance, oxidation, lipolysis, and thermogenesis. Loss of KDM4B in mice resulted in obesity associated with reduced energy expenditure and impaired adaptive thermogenesis. Obesity in KDM4B-deficient mice was accompanied by hyperlipidemia, insulin resistance, and pathological changes in the liver and pancreas. Adipocyte-specific deletion of Kdm4b revealed that the adipose tissues were the main sites for KDM4B antiobesity effects. KDM4B directly controlled the expression of multiple metabolic genes, including Ppargc1a and Ppara Collectively, our studies identify KDM4B as an essential epigenetic factor for the regulation of metabolic health and maintaining normal body weight in mice. KDM4B may provide a therapeutic target for treatment of obesity.

Regulation of hepatic autophagy by stress-sensing transcription factor CREBH.

Autophagy, a lysosomal degradative pathway in response to nutrient limitation, plays an important regulatory role in lipid homeostasis upon energy demands. Here, we demonstrated that the endoplasmic reticulum-tethered, stress-sensing transcription factor cAMP-responsive element-binding protein, hepatic-specific (CREBH) functions as a major transcriptional regulator of hepatic autophagy and lysosomal biogenesis in response to nutritional or circadian signals. CREBH deficiency led to decreased hepatic autophagic activities and increased hepatic lipid accumulation upon starvation. Under unfed or during energy-demanding phases of the circadian cycle, CREBH is activated to drive expression of the genes encoding the key enzymes or regulators in autophagosome formation or autophagic process, including microtubule-associated protein 1B-light chain 3, autophagy-related protein (ATG)7, ATG2b, and autophagosome formation Unc-51 like kinase 1, and the genes encoding functions in lysosomal biogenesis and homeostasis. Upon nutrient starvation, CREBH regulates and interacts with peroxisome proliferator-activated receptor α (PPARα) and PPARγ coactivator 1α to synergistically drive expression of the key autophagy genes and transcription factor EB, a master regulator of lysosomal biogenesis. Furthermore, CREBH regulates rhythmic expression of the key autophagy genes in the liver in a circadian-dependent manner. In summary, we identified CREBH as a key transcriptional regulator of hepatic autophagy and lysosomal biogenesis for the purpose of maintaining hepatic lipid homeostasis under nutritional stress or circadian oscillation.-Kim, H., Williams, D., Qiu, Y., Song, Z., Yang, Z., Kimler, V., Goldberg, A., Zhang, R., Yang, Z., Chen, X., Wang, L., Fang, D., Lin, J. D., Zhang, K. Regulation of hepatic autophagy by stress-sensing transcription factor CREBH.

Zbtb7b engages the long noncoding RNA Blnc1 to drive brown and beige fat development and thermogenesis.

Brown and beige adipocytes convert chemical energy into heat through uncoupled respiration to defend against cold stress. Beyond thermogenesis, brown and beige fats engage other metabolic tissues via secreted factors to influence systemic energy metabolism. How the protein and long noncoding RNA (lncRNA) regulatory networks act in concert to regulate key aspects of thermogenic adipocyte biology remains largely unknown. Here we developed a genome-wide functional screen to interrogate the transcription factors and cofactors in thermogenic gene activation and identified zinc finger and BTB domain-containing 7b (Zbtb7b) as a potent driver of brown fat development and thermogenesis and cold-induced beige fat formation. Zbtb7b is required for activation of the thermogenic gene program in brown and beige adipocytes. Genetic ablation of Zbtb7b impaired cold-induced transcriptional remodeling in brown fat, rendering mice sensitive to cold temperature, and diminished browning of inguinal white fat. Proteomic analysis revealed a mechanistic link between Zbtb7b and the lncRNA regulatory pathway through which Zbtb7b recruits the brown fat lncRNA 1 (Blnc1)/heterogeneous nuclear ribonucleoprotein U (hnRNPU) ribonucleoprotein complex to activate thermogenic gene expression in adipocytes. These findings illustrate the emerging concept of a protein-lncRNA regulatory network in the control of adipose tissue biology and energy metabolism.

Long Noncoding RNAs: A New Regulatory Code in Metabolic Control.

Long noncoding RNAs (lncRNAs) are emerging as an integral part of the regulatory information encoded in the genome. lncRNAs possess the unique capability to interact with nucleic acids and proteins, and exert discrete effects on numerous biological processes. Recent studies have delineated multiple lncRNA pathways that control metabolic tissue development and function. The expansion of the regulatory code that links nutrient and hormonal signals to tissue metabolism gives new insights into the genetic and pathogenic mechanisms underlying metabolic disease. This review discusses lncRNA biology with a focus on their role in the development, signaling, and function of key metabolic tissues.

Bmal1 in Perivascular Adipose Tissue Regulates Resting-Phase Blood Pressure Through Transcriptional Regulation of Angiotensinogen.

BACKGROUND: The perivascular adipose tissue (PVAT) surrounding vessels constitutes a distinct functional integral layer of the vasculature required to preserve vascular tone under physiological conditions. However, there is little information on the relationship between PVAT and blood pressure regulation, including its potential contributions to circadian blood pressure variation. METHODS: Using unique brown adipocyte-specific aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and angiotensinogen knockout mice, we determined the vasoactivity of homogenized PVAT in aortic rings and how brown adipocyte peripheral expression of Bmal1 and angiotensinogen in PVAT regulates the amplitude of diurnal change in blood pressure in mice. RESULTS: We uncovered a peripheral clock in PVAT and demonstrated that loss of Bmal1 in PVAT reduces blood pressure in mice during the resting phase, leading to a superdipper phenotype. PVAT extracts from wild-type mice significantly induced contractility of isolated aortic rings in vitro in an endothelium-independent manner. This property was impaired in PVAT from brown adipocyte-selective Bmal1-deficient (BA-Bmal1-KO) mice. The PVAT contractile properties were mediated by local angiotensin II, operating through angiotensin II type 1 receptor-dependent signaling in the isolated vessels and linked to PVAT circadian regulation of angiotensinogen. Indeed, angiotensinogen mRNA and angiotensin II levels in PVAT of BA-Bmal1-KO mice were significantly reduced. Systemic infusion of angiotensin II, in turn, reduced Bmal1 expression in PVAT while eliminating the hypotensive phenotype during the resting phase in BA-Bmal1-KO mice. Angiotensinogen, highly expressed in PVAT, shows circadian expression in PVAT, and selective deletion of angiotensinogen in brown adipocytes recapitulates the phenotype of selective deletion of Bmal1 in brown adipocytes. Furthermore, angiotensinogen is a transcriptional target of Bmal1 in PVAT. CONCLUSIONS: These data indicate that local Bmal1 in PVAT regulates angiotensinogen expression and the ensuing increase in angiotensin II, which acts on smooth muscle cells in the vessel walls to regulate vasoactivity and blood pressure in a circadian fashion during the resting phase. These findings will contribute to a better understanding of the cardiovascular complications of circadian disorders, alterations in the circadian dipping phenotype, and cross-talk between systemic and peripheral regulation of blood pressure.

The Liver Clock Controls Cholesterol Homeostasis through Trib1 Protein-mediated Regulation of PCSK9/Low Density Lipoprotein Receptor (LDLR) Axis.

Disruption of the body clock has been recognized as a risk factor for cardiovascular disease. How the circadian pacemaker interacts with the genetic factors associated with plasma lipid traits remains poorly understood. Recent genome-wide association studies have identified an expanding list of genetic variants that influence plasma cholesterol and triglyceride levels. Here we analyzed circadian regulation of lipid-associated candidate genes in the liver and identified two distinct groups exhibiting rhythmic and non-rhythmic patterns of expression during light-dark cycles. Liver-specific inactivation of Bmal1 led to elevated plasma LDL/VLDL cholesterol levels as a consequence of the disruption of the PCSK9/LDL receptor regulatory axis. Ablation of the liver clock perturbed diurnal regulation of lipid-associated genes in the liver and markedly reduced the expression of the non-rhythmically expressed gene Trib1. Adenovirus-mediated rescue of Trib1 expression lowered plasma PCSK9 levels, increased LDL receptor protein expression, and restored plasma cholesterol homeostasis in mice lacking a functional liver clock. These results illustrate an unexpected mechanism through which the biological clock regulates cholesterol homeostasis through its regulation of non-rhythmic genes in the liver.

Temporal orchestration of circadian autophagy rhythm by C/EBPß.

Temporal organization of tissue metabolism is important for maintaining nutrient and energy homeostasis in mammals. Autophagy is a conserved cellular pathway that is activated in response to nutrient limitation, resulting in the degradation of cytoplasmic components and the release of amino acids and other nutrients. Here, we show that autophagy exhibits robust circadian rhythm in mouse liver, which is accompanied by cyclic induction of genes involved in various steps of autophagy. Functional analyses of transcription factors and cofactors identified C/EBPß as a potent activator of autophagy. C/EBPß is rhythmically expressed in the liver and is regulated by both circadian and nutritional signals. In cultured primary hepatocytes, C/EBPß stimulates the program of autophagy gene expression and is sufficient to activate autophagic protein degradation. Adenoviral-mediated RNAi knockdown of C/EBPß in vivo abolishes diurnal autophagy rhythm in the liver. Further, circadian regulation of C/EBPß and autophagy is disrupted in mice lacking a functional liver clock. We have thus identified C/EBPß as a key factor that links autophagy to biological clock and maintains nutrient homeostasis throughout light/dark cycles.