Perturbed liver gene zonation in a mouse model of non-alcoholic steatohepatitis.

Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal hepatic function. Wnt signaling is a master regulator of spatial liver zonation. A perivenous-periportal Wnt activity gradient orchestrates metabolic zonation by activating gene expression in perivenous hepatocytes, while suppressing gene expression in their periportal counterparts. However, the understanding as to the liver gene zonation and zonation regulators in diseases is limited. Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fat accumulation, inflammation, and fibrosis. Here, we investigated the perturbation of liver gene zonation in a mouse NASH model by combining spatial transcriptomics, bulk RNAseq and in situ hybridization. Wnt-target genes represented a major subset of genes showing altered spatial expression in the NASH liver. The altered Wnt-target gene expression levels and zonation spatial patterns were in line with the up regulation of Wnt regulators and the augmentation of Wnt signaling. Particularly, we found that the Wnt activator Rspo3 expression was restricted to the perivenous zone in control liver but expanded to the periportal zone in NASH liver. AAV8-mediated RSPO3 overexpression in controls resulted in zonation changes, and further amplified the disturbed zonation of Wnt-target genes in NASH, similarly Rspo3 knockdown in Rspo3+/- mice resulted in zonation changes of Wnt-target genes in both chow and HFD mouse. Interestingly, there were no impacts on steatosis, inflammation, or fibrosis NASH pathology from RSPO3 overexpression nor Rspo3 knockdown. In summary, our study demonstrated the alteration of Wnt signaling in a mouse NASH model, leading to perturbed liver zonation.

Inducible hepatic expression of CREBH mitigates diet-induced obesity, insulin resistance, and hepatic steatosis in mice.

Cyclic AMP-responsive element-binding protein H (CREBH encoded by Creb3l3) is a transcription factor that regulates the expression of genes that control lipid and glucose metabolism as well as inflammation. CREBH is upregulated in the liver under conditions of overnutrition, and mice globally lacking the gene (CREBH-/-) are highly susceptible to diet-induced obesity, insulin resistance, and hepatic steatosis. The net protective effects of CREBH have been attributed in large part to the activities of fibroblast growth factor (Fgf)-21 (Fgf21), a target gene that promotes weight loss, improves glucose homeostasis, and reduces hepatic lipid accumulation. To explore the possibility that activation of the CREBH-Fgf21 axis could ameliorate established effects of high-fat feeding, we generated an inducible transgenic hepatocyte-specific CREBH overexpression mouse model (Tg-rtTA). Acute overexpression of CREBH in livers of Tg-rtTA mice effectively reversed diet-induced obesity, insulin resistance, and hepatic steatosis. These changes were associated with increased activities of thermogenic brown and beige adipose tissues in Tg-rtTA mice, leading to reductions in fat mass, along with enhanced insulin sensitivity and glucose tolerance. Genetically silencing Fgf21 in Tg-rtTA mice abrogated the CREBH-mediated reductions in body weight loss, but only partially reversed the observed improvements in glucose metabolism. These findings reveal that the protective effects of CREBH activation may be leveraged to mitigate diet-induced obesity and associated metabolic abnormalities in both Fgf21-dependent and Fgf21-independent pathways.

The Differential Expression of Cide Family Members is Associated with Nafld Progression from Steatosis to Steatohepatitis.

Improved understanding of the molecular mechanisms responsible for the progression from a "non-pathogenic" steatotic state to Non-Alcoholic Steatohepatitis is an important clinical requirement. The cell death-inducing DFF45 like effector (CIDE) family members (A, B and FSP27) regulate hepatic lipid homeostasis by controlling lipid droplet growth and/or VLDL production. However, CIDE proteins, particularly FSP27, have a dual role in that they also regulate cell death. We here report that the hepatic expression of CIDEA and FSP27 (α/ß) was similarly upregulated in a dietary mouse model of obesity-mediated hepatic steatosis. In contrast, CIDEA expression decreased, but FSP27-ß expression strongly increased in a dietary mouse model of steatohepatitis. The inverse expression pattern of CIDEA and FSP27ß was amplified with the increasing severity of the liver inflammation and injury. In obese patients, the hepatic CIDEC2 (human homologue of mouse FSP27ß) expression strongly correlated with the NAFLD activity score and liver injury. The hepatic expression of CIDEA tended to increase with obesity, but decreased with NAFLD severity. In hepatic cell lines, the downregulation of FSP27ß resulted in the fractionation of lipid droplets, whereas its overexpression decreased the expression of the anti-apoptotic BCL2 marker. This, in turn, sensitized cells to apoptosis in response to TNF α and saturated fatty acid. Considered together, our animal, human and in vitro studies indicate that differential expression of FSP27ß/CIDEC2 and CIDEA is related to NAFLD progression and liver injury.

Transcriptional profiling of PPARα-/- and CREB3L3-/- livers reveals disparate regulation of hepatoproliferative and metabolic functions of PPARα.

BACKGROUND: Peroxisome Proliferator-Activated receptor α (PPARα) and cAMP-Responsive Element Binding Protein 3-Like 3 (CREB3L3) are transcription factors involved in the regulation of lipid metabolism in the liver. The aim of the present study was to characterize the interrelationship between PPARα and CREB3L3 in regulating hepatic gene expression. Male wild-type, PPARα-/-, CREB3L3-/- and combined PPARα/CREB3L3-/- mice were subjected to a 16-h fast or 4 days of ketogenic diet. Whole genome expression analysis was performed on liver samples. RESULTS: Under conditions of overnight fasting, the effects of PPARα ablation and CREB3L3 ablation on plasma triglyceride, plasma ß-hydroxybutyrate, and hepatic gene expression were largely disparate, and showed only limited interdependence. Gene and pathway analysis underscored the importance of CREB3L3 in regulating (apo)lipoprotein metabolism, and of PPARα as master regulator of intracellular lipid metabolism. A small number of genes, including Fgf21 and Mfsd2a, were under dual control of PPARα and CREB3L3. By contrast, a strong interaction between PPARα and CREB3L3 ablation was observed during ketogenic diet feeding. Specifically, the pronounced effects of CREB3L3 ablation on liver damage and hepatic gene expression during ketogenic diet were almost completely abolished by the simultaneous ablation of PPARα. Loss of CREB3L3 influenced PPARα signalling in two major ways. Firstly, it reduced expression of PPARα and its target genes involved in fatty acid oxidation and ketogenesis. In stark contrast, the hepatoproliferative function of PPARα was markedly activated by loss of CREB3L3. CONCLUSIONS: These data indicate that CREB3L3 ablation uncouples the hepatoproliferative and lipid metabolic effects of PPARα. Overall, except for the shared regulation of a very limited number of genes, the roles of PPARα and CREB3L3 in hepatic lipid metabolism are clearly distinct and are highly dependent on dietary status.

Spliced XBP1 Rescues Renal Interstitial Inflammation Due to Loss of Sec63 in Collecting Ducts.

BACKGROUND: SEC63 encodes a resident protein in the endoplasmic reticulum membrane that, when mutated, causes human autosomal dominant polycystic liver disease. Selective inactivation of Sec63 in all distal nephron segments in embryonic mouse kidney results in polycystin-1-mediated polycystic kidney disease (PKD). It also activates the Ire1α-Xbp1 branch of the unfolded protein response, producing Xbp1s, the active transcription factor promoting expression of specific genes to alleviate endoplasmic reticulum stress. Simultaneous inactivation of Xbp1 and Sec63 worsens PKD in this model. METHODS: We explored the renal effects of postnatal inactivation of Sec63 alone or with concomitant inactivation of Xbp1 or Ire1α, specifically in the collecting ducts of neonatal mice. RESULTS: The later onset of inactivation of Sec63 restricted to the collecting duct does not result in overt activation of the Ire1α-Xbp1 pathway or cause polycystin-1-dependent PKD. Inactivating Sec63 along with either Xbp1 or Ire1α in this model causes interstitial inflammation and associated fibrosis with decline in kidney function over several months. Re-expression of XBP1s in vivo completely rescues the chronic kidney injury observed after inactivation of Sec63 with either Xbp1 or Ire1α. CONCLUSIONS: In the absence of Sec63, basal levels of Xbp1s activity in collecting ducts is both necessary and sufficient to maintain proteostasis (protein homeostasis) and protect against inflammation, myofibroblast activation, and kidney functional decline. The Sec63-Xbp1 double knockout mouse offers a novel genetic model of chronic tubulointerstitial kidney injury, using collecting duct proteostasis defects as a platform for discovery of signals that may underlie CKD of disparate etiologies.

A negative feedback loop between XBP1 and Fbw7 regulates cancer development.

In cancer, activation of X-box binding protein (XBP1) has a critical role in tumorigenesis and cancer progression. Transcriptional regulatory mechanism of XBP1 in cancer development has been well known, however, regulation of ubiquitination and degradation of XBP1 has not been elucidated yet. Here we show that Fbw7, a substrate recognition component of the SKP1-Cullin-F-box-type E3 ligase, interacts with XBP1 in a phosphorylation-dependent manner, and facilitates XBP1 ubiquitination and protein degradation. Moreover, Fbw7 inhibits oncogenic pathways including NF-κB, AP1, and Myc induced by XBP1. Interestingly, XBP1 negatively regulates transcription of Fbw7 via a feedback mechanism through NF-κB/E2F-1 axis signaling pathway, suggesting that overexpression of XBP1s may contribute to low level of Fbw7 expression in human cancers. Therefore, a negative feedback loop between Fbw7 and XBP1 contributes to the regulation of tumor development and can be an attractive target for novel therapy in cancers.

Gene Signature of the Human Pancreatic ε Cell.

The ghrelin-producing ε cell represents the fifth endocrine cell type in human pancreatic islets. The abundance of ε cells in adult pancreas is extremely low, which has hampered the investigation on the molecular pathways regulating the development and the function of this cell type. In this study, we explored the molecular features defining the function of pancreatic ε cells isolated from adult nondiabetic donors using single-cell RNA sequencing technology. We focus on transcription factors, cell surface receptors, and genes involved in metabolic pathways that contribute to regulation of cellular function. Furthermore, the genes that separate ε cells from the other islet endocrine cell types are presented. This study expands prior knowledge about the genes important for ε cell functioning during development and provides a resource to interrogate the transcriptome of this rare human islet cell type.

XBP1-KLF9 Axis Acts as a Molecular Rheostat to Control the Transition from Adaptive to Cytotoxic Unfolded Protein Response.

Transcription factor XBP1s, activated by endoplasmic reticulum (ER) stress in a dose-dependent manner, plays a central role in adaptive unfolded protein response (UPR) via direct activation of multiple genes controlling protein refolding. Here, we report that elevation of ER stress above a critical threshold causes accumulation of XBP1s protein sufficient for binding to the promoter and activation of a gene encoding a transcription factor KLF9. In comparison to other XBP1s targets, KLF9 promoter contains an evolutionary conserved lower-affinity binding site that requires higher amounts of XBP1s for activation. In turn, KLF9 induces expression of two regulators of ER calcium storage, TMEM38B and ITPR1, facilitating additional calcium release from ER, exacerbation of ER stress, and cell death. Accordingly, Klf9 deficiency attenuates tunicamycin-induced ER stress in mouse liver. These data reveal a role for XBP1s in cytotoxic UPR and provide insights into mechanisms of life-or-death decisions in cells under ER stress.

Gene Signature of Proliferating Human Pancreatic α Cells.

Pancreatic α cells proliferate at a low rate, and little is known about the control of this process. Here we report the characterization of human α cells by large-scale, single-cell RNA sequencing coupled with pseudotime ordering. We identified two large subpopulations and a smaller cluster of proliferating α cells with increased expression of genes involved in cell-cycle regulation. The proliferating α cells were differentiated, had normal levels of GCG expression, and showed no signs of cellular stress. Proliferating α cells were detected in both the G1S and G2M phases of the cell cycle. Human α cells proliferate at a fivefold higher rate than human ß cells and express lower levels of the cell-cycle inhibitors CDKN1A and CDKN1C. Collectively, this study provides the gene signatures of human α cells and the genes involved in their cell division. The lower expression of two cell-cycle inhibitors in human α cells could account for their higher rate of proliferation compared with their insulin-producing counterparts.

Pseudotime Ordering of Single Human ß-Cells Reveals States of Insulin Production and Unfolded Protein Response.

Proinsulin is a misfolding-prone protein, making its biosynthesis in the endoplasmic reticulum (ER) a stressful event. Pancreatic ß-cells overcome ER stress by activating the unfolded protein response (UPR) and reducing insulin production. This suggests that ß-cells transition between periods of high insulin biosynthesis and UPR-mediated recovery from cellular stress. We now report the pseudotime ordering of single ß-cells from humans without diabetes detected by large-scale RNA sequencing. We identified major states with 1) low UPR and low insulin gene expression, 2) low UPR and high insulin gene expression, or 3) high UPR and low insulin gene expression. The latter state was enriched for proliferating cells. Stressed human ß-cells do not dedifferentiate and show little propensity for apoptosis. These data suggest that human ß-cells transition between states with high rates of biosynthesis to fulfill the body's insulin requirements to maintain normal blood glucose levels and UPR-mediated recovery from ER stress due to high insulin production.

Preemptive Activation of the Integrated Stress Response Protects Mice From Diet-Induced Obesity and Insulin Resistance by Fibroblast Growth Factor 21 Induction.

Integrated stress response (ISR) is a signaling system in which phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by stress-specific kinases and subsequent activation of activation transcription factor (ATF) 4 help restore cellular homeostasis following exposure to environmental stresses. ISR activation has been observed in metabolic diseases, including hepatic steatosis (HS), steatohepatitis (SH), and insulin resistance (IR), but it remains unclear whether ISR contributes to disease pathogenesis or represents an innate defense mechanism against metabolic stresses. Constitutive repressor of eIF2α phosphorylation (CReP) is a critical regulatory subunit of the eIF2α phosphatase complex. Here, we show that CReP ablation causes constitutive eIF2α phosphorylation in the liver, which leads to activation of the ATF4 transcriptional program including increased fibroblast growth factor 21 (FGF21) production. Liver-specific CReP knockout (CRePLKO ) mice exhibited marked browning of white adipose tissue (WAT) and increased energy expenditure and insulin sensitivity in an FGF21-dependent manner. Furthermore, CRePLKO mice were protected from high-fat diet (HFD)-induced obesity, HS, and IR. Acute CReP ablation in liver of HFD-induced obese mice also reduced adiposity and improved glucose homeostasis. Conclusion: These data suggest that CReP abundance is a critical determinant for eIF2α phosphorylation and ensuing ISR activation in the liver. Constitutive ISR activation in the liver induces FGF21 and confers protection from HFD-induced adiposity, IR, and HS in mice. Augmenting hepatic ISR may represent a therapeutic approach to treat metabolic disorders.

Very Low Density Lipoprotein Assembly Is Required for cAMP-responsive Element-binding Protein H Processing and Hepatic Apolipoprotein A-IV Expression.

Hepatic apolipoprotein A-IV (apoA-IV) expression is correlated with hepatic triglyceride (TG) content in mouse models of chronic hepatosteatosis, and steatosis-induced hepatic apoA-IV gene expression is regulated by nuclear transcription factor cAMP-responsive element-binding protein H (CREBH) processing. To define what aspects of TG homeostasis regulate hepatic CREBH processing and apoA-IV gene expression, several mouse models of attenuated VLDL particle assembly were subjected to acute hepatosteatosis induced by an overnight fast or short term ketogenic diet feeding. Compared with chow-fed C57BL/6 mice, fasted or ketogenic diet-fed mice displayed increased hepatic TG content, which was highly correlated (r2 = 0.95) with apoA-IV gene expression, and secretion of larger, TG-enriched VLDL, despite a lower rate of TG secretion and a similar or reduced rate of apoB100 secretion. When VLDL particle assembly and secretion was inhibited by hepatic shRNA-induced apoB silencing or genetic or pharmacologic reduction in microsomal triglyceride transfer protein (MTP) activity, hepatic TG content increased dramatically; however, CREBH processing and apoA-IV gene expression were attenuated compared with controls. Adenovirus-mediated reconstitution of MTP expression proportionately restored CREBH processing and apoA-IV expression in liver-specific MTP knock-out mice. These results reveal that hepatic TG content, per se, does not regulate CREBH processing. Instead, TG mobilization into the endoplasmic reticulum for nascent VLDL particle assembly activates CREBH processing and enhances apoA-IV gene expression in the setting of acute steatosis. We conclude that VLDL assembly and CREBH activation play key roles in the response to hepatic steatosis by up-regulating apoA-IV and promoting assembly and secretion of larger, more TG-enriched VLDL particles.

MIST1 Links Secretion and Stress as both Target and Regulator of the Unfolded Protein Response.

Transcriptional networks that govern secretory cell specialization, including instructing cells to develop a unique cytoarchitecture, amass extensive protein synthesis machinery, and be embodied to respond to endoplasmic reticulum (ER) stress, remain largely uncharacterized. In this study, we discovered that the secretory cell transcription factor MIST1 (Bhlha15), previously shown to be essential for cytoskeletal organization and secretory activity, also functions as a potent ER stress-inducible transcriptional regulator. Genome-wide DNA binding studies, coupled with genetic mouse models, revealed MIST1 gene targets that function along the entire breadth of the protein synthesis, processing, transport, and exocytosis networks. Additionally, key MIST1 targets are essential for alleviating ER stress in these highly specialized cells. Indeed, MIST1 functions as a coregulator of the unfolded protein response (UPR) master transcription factor XBP1 for a portion of target genes that contain adjacent MIST1 and XBP1 binding sites. Interestingly, Mist1 gene expression is induced during ER stress by XBP1, but as ER stress subsides, MIST1 serves as a feedback inhibitor, directly binding the Xbp1 promoter and repressing Xbp1 transcript production. Together, our findings provide a new paradigm for XBP1-dependent UPR regulation and position MIST1 as a potential biotherapeutic for numerous human diseases.

Loss of Transcription Factor CREBH Accelerates Diet-Induced Atherosclerosis in Ldlr-/- Mice.

OBJECTIVE: Liver-enriched transcription factor cAMP-responsive element-binding protein H (CREBH) regulates plasma triglyceride clearance by inducing lipoprotein lipase cofactors, such as apolipoprotein A-IV (apoA-IV), apoA-V, and apoC-II. CREBH also regulates apoA-I transcription. This study aims to determine whether CREBH has a role in lipoprotein metabolism and development of atherosclerosis. APPROACH AND RESULTS: CREBH-deficient Creb3l3(-/-) mice were bred with Ldlr(-/-) mice creating Ldlr(-/-) Creb3l3(-/-) double knockout mice. Mice were fed on a high-fat and high-sucrose Western diet for 20 weeks. We showed that CREBH deletion in Ldlr(-/-) mice increased very low-density lipoprotein-associated triglyceride and cholesterol levels, consistent with the impairment of lipoprotein lipase-mediated triglyceride clearance in these mice. In contrast, high-density lipoprotein cholesterol levels were decreased in CREBH-deficient mice, which was associated with decreased production of apoA-I from the liver. The results indicate that CREBH directly activated Apoa1 gene transcription. Accompanied by the worsened atherogenic lipid profile, Ldlr(-/-) Creb3l3(-/-) mice developed significantly more atherosclerotic lesions in the aortas than Ldlr(-/-) mice. CONCLUSIONS: We identified CREBH as an important regulator of lipoprotein metabolism and suggest that increasing hepatic CREBH activity may be a novel strategy for prevention and treatment of atherosclerosis.

CREBH-FGF21 axis improves hepatic steatosis by suppressing adipose tissue lipolysis.

Adipose tissue lipolysis produces glycerol and nonesterified fatty acids (NEFA) that serve as energy sources during nutrient scarcity. Adipose tissue lipolysis is tightly regulated and excessive lipolysis causes hepatic steatosis, as NEFA released from adipose tissue constitutes a major source of TG in the liver of patients with nonalcoholic fatty liver diseases. Here we show that the liver-enriched transcription factor CREBH is activated by TG accumulation and induces FGF21, which suppresses adipose tissue lipolysis, ameliorating hepatic steatosis. CREBH-deficient mice developed severe hepatic steatosis due to increased adipose tissue lipolysis, when fasted or fed a high-fat low-carbohydrate ketogenic diet. FGF21 production was impaired in CREBH-deficient mice, and adenoviral overexpression of FGF21 suppressed adipose tissue lipolysis and improved hepatic steatosis in these mice. Thus, our results uncover a negative feedback loop in which CREBH regulates NEFA flux from adipose tissue to the liver via FGF21.