Trans-omic analysis reveals opposite metabolic dysregulation between feeding and fasting in liver associated with obesity.

Dysregulation of liver metabolism associated with obesity during feeding and fasting leads to the breakdown of metabolic homeostasis. However, the underlying mechanism remains unknown. Here, we measured multi-omics data in the liver of wild-type and leptin-deficient obese (ob/ob) mice at ad libitum feeding and constructed a differential regulatory trans-omic network of metabolic reactions. We compared the trans-omic network at feeding with that at 16 h fasting constructed in our previous study. Intermediate metabolites in glycolytic and nucleotide metabolism decreased in ob/ob mice at feeding but increased at fasting. Allosteric regulation reversely shifted between feeding and fasting, generally showing activation at feeding while inhibition at fasting in ob/ob mice. Transcriptional regulation was similar between feeding and fasting, generally showing inhibiting transcription factor regulations and activating enzyme protein regulations in ob/ob mice. The opposite metabolic dysregulation between feeding and fasting characterizes breakdown of metabolic homeostasis associated with obesity.

An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

Hepatic FASN deficiency differentially affects nonalcoholic fatty liver disease and diabetes in mouse obesity models.

Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes are interacting comorbidities of obesity, and increased hepatic de novo lipogenesis (DNL), driven by hyperinsulinemia and carbohydrate overload, contributes to their pathogenesis. Fatty acid synthase (FASN), a key enzyme of hepatic DNL, is upregulated in association with insulin resistance. However, the therapeutic potential of targeting FASN in hepatocytes for obesity-associated metabolic diseases is unknown. Here, we show that hepatic FASN deficiency differentially affects NAFLD and diabetes depending on the etiology of obesity. Hepatocyte-specific ablation of FASN ameliorated NAFLD and diabetes in melanocortin 4 receptor-deficient mice but not in mice with diet-induced obesity. In leptin-deficient mice, FASN ablation alleviated hepatic steatosis and improved glucose tolerance but exacerbated fed hyperglycemia and liver dysfunction. The beneficial effects of hepatic FASN deficiency on NAFLD and glucose metabolism were associated with suppression of DNL and attenuation of gluconeogenesis and fatty acid oxidation, respectively. The exacerbation of fed hyperglycemia by FASN ablation in leptin-deficient mice appeared attributable to impairment of hepatic glucose uptake triggered by glycogen accumulation and citrate-mediated inhibition of glycolysis. Further investigation of the therapeutic potential of hepatic FASN inhibition for NAFLD and diabetes in humans should thus consider the etiology of obesity.

l-Type amino acid transporter 1 in hypothalamic neurons in mice maintains energy and bone homeostasis.

Hypothalamic neurons regulate body homeostasis by sensing and integrating changes in the levels of key hormones and primary nutrients (amino acids, glucose, and lipids). However, the molecular mechanisms that enable hypothalamic neurons to detect primary nutrients remain elusive. Here, we identified l-type amino acid transporter 1 (LAT1) in hypothalamic leptin receptor-expressing (LepR-expressing) neurons as being important for systemic energy and bone homeostasis. We observed LAT1-dependent amino acid uptake in the hypothalamus, which was compromised in a mouse model of obesity and diabetes. Mice lacking LAT1 (encoded by solute carrier transporter 7a5, Slc7a5) in LepR-expressing neurons exhibited obesity-related phenotypes and higher bone mass. Slc7a5 deficiency caused sympathetic dysfunction and leptin insensitivity in LepR-expressing neurons before obesity onset. Importantly, restoring Slc7a5 expression selectively in LepR-expressing ventromedial hypothalamus neurons rescued energy and bone homeostasis in mice deficient for Slc7a5 in LepR-expressing cells. Mechanistic target of rapamycin complex-1 (mTORC1) was found to be a crucial mediator of LAT1-dependent regulation of energy and bone homeostasis. These results suggest that the LAT1/mTORC1 axis in LepR-expressing neurons controls energy and bone homeostasis by fine-tuning sympathetic outflow, thus providing in vivo evidence of the implications of amino acid sensing by hypothalamic neurons in body homeostasis.

The transcription factor ATF3 switches cell death from apoptosis to necroptosis in hepatic steatosis in male mice.

Hepatocellular death increases with hepatic steatosis aggravation, although its regulation remains unclear. Here we show that hepatic steatosis aggravation shifts the hepatocellular death mode from apoptosis to necroptosis, causing increased hepatocellular death. Our results reveal that the transcription factor ATF3 acts as a master regulator in this shift by inducing expression of RIPK3, a regulator of necroptosis. In severe hepatic steatosis, after partial hepatectomy, hepatic ATF3-deficient or -overexpressing mice display decreased or increased RIPK3 expression and necroptosis, respectively. In cultured hepatocytes, ATF3 changes TNFα-dependent cell death mode from apoptosis to necroptosis, as revealed by live-cell imaging. In non-alcoholic steatohepatitis (NASH) mice, hepatic ATF3 deficiency suppresses RIPK3 expression and hepatocellular death. In human NASH, hepatocellular damage is correlated with the frequency of hepatocytes expressing ATF3 or RIPK3, which overlap frequently. ATF3-dependent RIPK3 induction, causing a modal shift of hepatocellular death, can be a therapeutic target for steatosis-induced liver damage, including NASH.

Diet intake control is indispensable for the gluconeogenic response to sodium-glucose cotransporter 2 inhibition in male mice.

AIMS/INTRODUCTION: Sodium-glucose cotransporter 2 inhibitor (SGLT2i) lowers blood glucose and causes a whole-body energy deficit by boosting renal glucose excretion, thus affecting glucose and energy metabolism. This energy deficit not only decreases bodyweight, but also increases food intake. This food intake increase offsets the SGLT2i-induced bodyweight decrease, but the effect of the food intake increase on the SGLT2i regulation of glucose metabolism remains unclear. MATERIALS AND METHODS: We administered SGLT2i (luseogliflozin) for 4 weeks to hepatic gluconeogenic enzyme gene G6pc reporter mice with/without obesity, which were either fed freely or under a 3-hourly dietary regimen. The effect of feeding condition on the gluconeogenic response to SGLT2i was evaluated by plasma Gaussia luciferase activity, an index of the hepatic gluconeogenic response, in G6pc reporter mice. Energy expenditure was measured by indirect calorimetry. RESULTS: In the lean mice under controlled feeding, SGLT2i decreased bodyweight and plasma glucose, and increased the hepatic gluconeogenic response while decreasing blood insulin. SGLT2i also increased oxygen consumption under controlled feeding. However, free feeding negated all of these effects of SGLT2i. In the obese mice, SGLT2i decreased bodyweight, blood glucose and plasma insulin, ameliorated the upregulated hepatic gluconeogenic response, and increased oxygen consumption under controlled feeding. Under free feeding, although blood glucose was decreased and plasma insulin tended to decrease, the effects of SGLT2i - decreased bodyweight, alleviation of the hepatic gluconeogenic response and increased oxygen consumption - were absent. CONCLUSIONS: Food intake management is crucial for SGLT2i to affect glucose and energy metabolism during type 2 diabetes treatment.

GCN2 regulates pancreatic ß cell mass by sensing intracellular amino acid levels.

EIF2AK4, which encodes the amino acid deficiency-sensing protein GCN2, has been implicated as a susceptibility gene for type 2 diabetes in the Japanese population. However, the mechanism by which GCN2 affects glucose homeostasis is unclear. Here, we show that insulin secretion is reduced in individuals harboring the risk allele of EIF2AK4 and that maintenance of GCN2-deficient mice on a high-fat diet results in a loss of pancreatic ß cell mass. Our data suggest that GCN2 senses amino acid deficiency in ß cells and limits signaling by mechanistic target of rapamycin complex 1 to prevent ß cell failure during the consumption of a high-fat diet.

Hepatic Gluconeogenic Response to Single and Long-Term SGLT2 Inhibition in Lean/Obese Male Hepatic G6pc-Reporter Mice.

Sodium-glucose cotransporter 2 inhibitor (SGLT2i) consistently reduces blood glucose levels in type 2 diabetes mellitus but increases hepatic gluconeogenic gene expression and glucose production, offsetting its glucose-lowering effect. This study aimed to elucidate the effect of SGLT2i on hepatic gluconeogenic response and its mechanism in both insulin-sensitive and insulin-resistant states. A hepatic mouse model was generated to show liver-specific expression of Gaussia luciferase (GLuc) driven by the gluconeogenic enzyme gene G6pc promoter. Hepatic gluconeogenic response was evaluated by measuring plasma GLuc activity. SGLT2i was given to lean and obese mice in single gavage administration or 4-week dietary administration with controlled feeding every 3 hours. In lean mice, single-dose SGLT2i increased plasma GLuc activity from 2 hours after administration, decreasing blood glucose and plasma insulin from 1 to 2 hours after administration. In obese mice, which had higher plasma GLuc activity than lean ones, SGLT2i did not further increase GLuc activity despite decreased blood glucose and plasma insulin. Hepatic Akt and GSK3ß phosphorylation was attenuated by single-dose SGLT2i in lean mice in accordance with the plasma insulin decrease, but not in obese mice. Long-term SGLT2i administration, which increased plasma GLuc activity in lean mice, decreased it in obese mice from 3 weeks after initiation, with increased hepatic Akt and GSK3ß phosphorylation. In conclusion, single SGLT2i administration increases hepatic gluconeogenic response in lean insulin-sensitive mice, but not in obese insulin-resistant mice. Long-term SGLT2i administration relieves obesity-induced upregulation of the hepatic gluconeogenic response by restoring impeded hepatic insulin signaling in obese insulin-resistant mice.

MAPK Erk5 in Leptin Receptor‒Expressing Neurons Controls Body Weight and Systemic Energy Homeostasis in Female Mice.

Extracellular signal-regulated kinase 5 (Erk5), a member of the MAPK family, is specifically phosphorylated and activated by MAPK/Erk kinase-5. Although it has been implicated in odor discrimination and long-term memory via its expression in the central nervous system, little is known regarding the physiological importance of neuronal Erk5 in body weight and energy homeostasis. In the current study, systemic insulin injection significantly induced phosphorylation of Erk5 in the hypothalamus. Moreover, Erk5 deficiency in leptin receptor (LepR)‒expressing neurons led to an obesity phenotype, with increased white adipose tissue mass due to increased adipocyte size, only in female mice fed a normal chow diet. Furthermore, Erk5 deficiency in LepR-expressing neurons showed impaired glucose tolerance along with decreased physical activity, food intake, and energy expenditure. These results suggest that Erk5 controls body weight and systemic energy homeostasis probably via its expression in hypothalamic neurons in female mice, thereby providing a target for metabolic diseases such as obesity and type 2 diabetes mellitus.

Nicotinic alpha-7 acetylcholine receptor deficiency exacerbates hepatic inflammation and fibrosis in a mouse model of non-alcoholic steatohepatitis.

AIMS/INTRODUCTION: Non-alcoholic steatohepatitis (NASH), which occurs in association with insulin resistance and hepatic fat accumulation, is characterized by chronic liver injury and fibrosis. NASH onset and progression is closely related to hepatic inflammation, which is partly regulated by the vagus nerve through the α7 nicotinic acetylcholine receptor (α7nAchR). Hepatic α7nAchR action is impeded in obesity and insulin resistance. In the present study, using α7nAchR knockout (α7KO) mice, we elucidated the effect of α7nAchR deficiency on NASH-related inflammation and fibrosis. MATERIALS AND METHODS: α7KO mice were fed an atherogenic high-fat diet (AD) for 32 weeks or methionine/choline-deficient diet (MCD) for 6 weeks, both of which induce NASH. Mice were then examined for the degree of NASH-related inflammation and fibrosis by hepatic gene expression analysis and Sirius red histological staining. RESULTS: Hepatic triglyceride accumulation and elevated plasma transaminase levels were observed in both AD and MCD mice, but the plasma transaminase level increase was higher in α7KO mice than in control mice. α7KO mice fed an AD showed significant upregulation of the Col1a1 gene encoding alpha-1 type I collagen, which is involved in liver fibrosis, and the Ccl2 gene encoding C-C motif chemokine ligand 2, a pro-inflammatory chemokine; α7KO mice fed an MCD had significant upregulation of the Col1a1 gene and the Tnf gene, an inflammatory cytokine. Histological analysis showed that AD and MCD exacerbated liver fibrosis in α7KO mice. CONCLUSIONS: The results of this study suggest that α7nAchR deficiency exacerbates hepatic inflammation and fibrosis in a diet-induced mouse model of NASH.

PHD3 regulates glucose metabolism by suppressing stress-induced signalling and optimising gluconeogenesis and insulin signalling in hepatocytes.

Glucagon-mediated gene transcription in the liver is critical for maintaining glucose homeostasis. Promoting the induction of gluconeogenic genes and blocking that of insulin receptor substrate (Irs)2 in hepatocytes contributes to the pathogenesis of type 2 diabetes. However, the molecular mechanism by which glucagon signalling regulates hepatocyte metabolism is not fully understood. We previously showed that a fasting-inducible signalling module consisting of general control non-repressed protein 5, co-regulator cAMP response element-binding protein binding protein/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2, and protein kinase A is required for glucagon-induced transcription of gluconeogenic genes. The present study aimed to identify the downstream effectors of this module in hepatocytes by examining glucagon-induced potential target genes. One of these genes was prolyl hydroxylase domain (PHD)3, which suppressed stress signalling through inhibition of the IκB kinase-nuclear factor-κB pathway in a proline hydroxylase-independent manner to maintain insulin signalling. PHD3 was also required for peroxisome proliferator-activated receptor γ coactivator 1α-induced gluconeogenesis, which was dependent on proline hydroxylase activity, suggesting that PHD3 regulates metabolism in response to glucagon as well as insulin. These findings demonstrate that glucagon-inducible PHD3 regulates glucose metabolism by suppressing stress signalling and optimising gluconeogenesis and insulin signalling in hepatocytes.

Dietary soybean protein ameliorates high-fat diet-induced obesity by modifying the gut microbiota-dependent biotransformation of bile acids.

The consumption of soybean protein has well-known favorable metabolic effects (e.g., reduced body weight, body fat, hyperglycemia, insulin resistance, hepatic steatosis, and lipogenesis). These effects of soy protein have been linked to modulation by the gut microbiota; however, the dynamic interplay among these factors remains unclear. Accordingly, we examined the metabolic phenotype, intestinal BA pool, and the gut microbiome of male C57BL/6 mice that were randomized to receive either a regular high-fat diet (HFD) or HFD that contained soybean protein isolate (SPI) in place of dairy protein. The intake of SPI significantly reduced the HFD-induced weight gain and adipose tissue mass accumulation and attenuated hepatic steatosis. Along with an enhancement in the secretion of intestinal Glucagon-like peptide-1 (GLP-1), an enlarged cecal BA pool with an elevated secondary/primary BA ratio was observed in the mice that consumed SPI, while fecal BA excretion remained unaltered. SPI also elicited dramatic changes in the gut microbiome, characterized by an expansion of taxa that may be involved in the biotransformation of BAs. The observed effects were abolished in germ-free (GF) mice, indicating that they were dependent on the microbiota. These findings collectively indicate that the metabolic benefits of SPI under the HFD regime may arise from a microbiota-driven increase in BA transformation and increase in GLP-1 secretion.

Sirt2 facilitates hepatic glucose uptake by deacetylating glucokinase regulatory protein.

Impaired hepatic glucose uptake (HGU) causes postprandial hyperglycemia in type 2 diabetes. Here, we show that diminished hepatic Sirt2 activity impairs HGU in obese diabetic mice. Hepatic Sirt2 overexpression increases HGU in high-fat diet (HFD)-fed obese diabetic mice and mitigates their impaired glucose tolerance. Hepatic Sirt2 knockdown in non-diabetic mice reduces HGU and causes impaired glucose tolerance. Sirt2 promotes glucose-dependent HGU by deacetylating K126 of glucokinase regulatory protein (GKRP). Glucokinase and GKRP glucose-dependent dissociation is necessary for HGU but is inhibited in hepatocytes derived from obese diabetic mice, depleted of Sirt2 or transfected with GKRP acetylation-mimicking mutants. GKRP deacetylation-mimicking mutants dissociate from glucokinase in a glucose concentration-dependent manner in obese diabetic mouse-derived hepatocytes and increase HGU and glucose tolerance in HFD-induced or db/db obese diabetic mice. We demonstrate that Sirt2-dependent GKRP deacetylation improves impaired HGU and suggest that it may be a therapeutic target for type 2 diabetes.

PDGFRß Regulates Adipose Tissue Expansion and Glucose Metabolism via Vascular Remodeling in Diet-Induced Obesity.

Platelet-derived growth factor (PDGF) is a key factor in angiogenesis; however, its role in adult obesity remains unclear. In order to clarify its pathophysiological role, we investigated the significance of PDGF receptor ß (PDGFRß) in adipose tissue expansion and glucose metabolism. Mature vessels in the epididymal white adipose tissue (eWAT) were tightly wrapped with pericytes in normal mice. Pericyte desorption from vessels and the subsequent proliferation of endothelial cells were markedly increased in the eWAT of diet-induced obese mice. Analyses with flow cytometry and adipose tissue cultures indicated that PDGF-B caused the detachment of pericytes from vessels in a concentration-dependent manner. M1-macrophages were a major type of cells expressing PDGF-B in obese adipose tissue. In contrast, pericyte detachment was attenuated and vascularity within eWAT was reduced in tamoxifen-inducible conditional Pdgfrb-knockout mice with decreases in adipocyte size and chronic inflammation. Furthermore, Pdgfrb-knockout mice showed enhanced energy expenditure. Consequently, diet-induced obesity and the associated deterioration of glucose metabolism in wild-type mice were absent in Pdgfrb-knockout mice. Therefore, PDGF-B-PDGFRß signaling plays a significant role in the development of adipose tissue neovascularization and appears to be a fundamental target for the prevention of obesity and type 2 diabetes.

Dietary Mung Bean Protein Reduces Hepatic Steatosis, Fibrosis, and Inflammation in Male Mice with Diet-Induced, Nonalcoholic Fatty Liver Disease.

BACKGROUND: As the prevalence of nonalcoholic fatty liver disease (NAFLD), including steatosis and nonalcoholic steatohepatitis, is increasing, novel dietary approaches are required for the prevention and treatment of NAFLD. OBJECTIVE: We evaluated the potential of mung bean protein isolate (MuPI) to prevent NAFLD progression. METHODS: In Expts. 1 and 2, the hepatic triglyceride (TG) concentration was compared between 8-wk-old male mice fed a high-fat diet (61% of energy from fat) containing casein, MuPI, and soy protein isolate and an MuPI-constituent amino acid mixture as a source of amino acids (18% of energy) for 4 wk. In Expt. 3, hepatic fatty acid synthase (Fasn) expression was evaluated in 8-wk-old male Fasn-promoter-reporter mice fed a casein- or MuPI-containing high-fat diet for 20 wk. In Expt. 4, hepatic fibrosis was examined in 8-wk-old male mice fed an atherogenic diet (61% of energy from fat, containing 1.3 g cholesterol/100 g diet) containing casein or MuPI (18% of energy) as a protein source for 20 wk. RESULTS: In the high fat-diet mice, the hepatic TG concentration in the MuPI group decreased by 66% and 47% in Expt. 1 compared with the casein group (P < 0.001) and the soy protein isolate group (P = 0.001), respectively, and decreased by 56% in Expt. 2 compared with the casein group (P = 0.011). However, there was no difference between the MuPI-constituent amino acid mixture and casein groups in Expt. 2. In Expt. 3, Fasn-promoter-reporter activity and hepatic TG concentration were lower in the MuPI group than in those fed casein (P < 0.05). In Expt. 4, in mice fed an atherogenic diet, hepatic fibrosis was not induced in the MuPI group, whereas it developed overtly in the casein group. CONCLUSION: MuPI potently reduced hepatic lipid accumulation in mice and may be a potential foodstuff to prevent NAFLD onset and progression.

Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor.

Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity.

Fine tuning of agonistic/antagonistic activity for vitamin D receptor by 22-alkyl chain length of ligands: 22S-Hexyl compound unexpectedly restored agonistic activity.

1α,25-Dihydroxyvitamin D3 exerts its actions by binding to vitamin D receptor (VDR). We are continuing the study related to the alteration of pocket structure of VDR by 22-alkyl substituent of ligands and the relationships between the alteration and agonistic/antagonistic activity. Previously we reported that compounds 2 (22-H), 3 (22S-Et), and 4 (22S-Bu) are VDR agonist, partial agonist and antagonist, respectively. Here, we describe the synthesis and biological evaluation of 22S-hexyl analog 5 (22S-Hex), which was designed to be a stronger VDR antagonist than 4. Unexpectedly, 5 showed partial agonistic but not antagonistic activity when bound to VDR, indicating that it is not necessarily true that the bulkier the side chain is, the stronger the antagonistic activity will be. X-ray crystallographic analysis of the VDR-ligand-binding domain (VDR-LBD) accommodating compound 5 indicated that the partial agonist activity of 5 is dependent on the mixed population of the agonistic and antagonistic conformations. Binding of compound 5 may not bring the complex into the only antagonistic conformation due to the large conformational change of the VDR-LBD. From this study it was found that fine tuning of agonistic/antagonistic activity for VDR is possible by 22-alkyl chain length of ligands.

Chronic activation of FXR in transgenic mice caused perinatal toxicity and sensitized mice to cholesterol toxicity.

The nuclear receptor farnesoid X receptor (FXR) (nuclear receptor subfamily 1, group H, member 4, or NR1H4) is highly expressed in the liver and intestine. Previous reports have suggested beneficial functions of FXR in the homeostasis of bile acids, lipids, and glucose, as well as in promoting liver regeneration and inhibiting carcinogenesis. To investigate the effect of chronic FXR activation in vivo, we generated transgenic mice that conditionally and tissue specifically express the activated form of FXR in the liver and intestine. Unexpectedly, the transgenic mice showed several intriguing phenotypes, including partial neonatal lethality, growth retardation, and spontaneous liver toxicity. The transgenic mice also displayed heightened sensitivity to a high-cholesterol diet-induced hepatotoxicity but resistance to the gallstone formation. The phenotypes were transgene specific, because they were abolished upon treatment with doxycycline to silence the transgene expression. The perinatal toxicity, which can be rescued by a maternal vitamin supplement, may have resulted from vitamin deficiency due to low biliary bile acid output as a consequence of inhibition of bile acid formation. Our results also suggested that the fibroblast growth factor-inducible immediate-early response protein 14 (Fn14), a member of the proinflammatory TNF family, is a FXR-responsive gene. However, the contribution of Fn14 induction in the perinatal toxic phenotype of the transgenic mice remains to be defined. Because FXR is being explored as a therapeutic target, our results suggested that a chronic activation of this nuclear receptor may have an unintended side effect especially during the perinatal stage.

Growth arrest and DNA damage-inducible 34 regulates liver regeneration in hepatic steatosis in mice.

UNLABELLED: The liver has robust regenerative potential in response to damage, but hepatic steatosis (HS) weakens this potential. We found that the enhanced integrated stress response (ISR) mediated by phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) impairs regeneration in HS and that growth arrest and DNA damage-inducible 34 (Gadd34)-dependent suppression of ISR plays a crucial role in fatty liver regeneration. Although mice fed a high-fat diet for 2 weeks developed moderate fatty liver with no increase in eIF2α phosphorylation before 70% hepatectomy, they showed impaired liver regeneration as a result of reduced proliferation and increased death of hepatocytes with increased phosphorylation of eIF2α and ISR. An increased ISR through Gadd34 knockdown induced C/EBP homologous protein (CHOP)-dependent apoptosis and receptor-interacting protein kinase 3-dependent necrosis, resulting in increased hepatocyte death during fatty liver regeneration. Furthermore, Gadd34 knockdown and increased phosphorylation of eIF2α decreased cyclin D1 protein and reduced hepatocyte proliferation. In contrast, enhancement of Gadd34 suppressed phosphorylation of eIF2α and reduced CHOP expression and hepatocyte apoptosis without affecting hepatocyte proliferation, clearly improving fatty liver regeneration. In more severe fatty liver of leptin receptor-deficient db/db mice, forced expression of hepatic Gadd34 also promoted hepatic regeneration after hepatectomy. CONCLUSION: Gadd34-mediated regulation of ISR acts as a physiological defense mechanism against impaired liver regeneration resulting from steatosis and is thus a possible therapeutic target for impaired regeneration in HS.

p62/SQSTM1 plays a protective role in oxidative injury of steatotic liver in a mouse hepatectomy model.

AIMS: Liver injury and regeneration involve complicated processes and are affected by various physio-pathological factors. We investigated the mechanisms of steatosis-associated liver injury and delayed regeneration in a mouse model of partial hepatectomy. RESULTS: Initial regeneration of the steatotic liver was significantly delayed after hepatectomy. Although hepatocyte proliferation was not significantly suppressed, severe liver injury with oxidative stress (OS) occurred immediately after hepatectomy in the steatotic liver. Fas-ligand (FasL)/Fas expression was upregulated in the steatotic liver, whereas the expression of antioxidant and anti-apoptotic molecules (catalase/MnSOD/Ref-1 and Bcl-2/Bcl-xL/FLIP, respectively) and p62/SQSTM1, a steatosis-associated protein, was downregulated. Interestingly, pro-survival Akt was not activated in response to hepatectomy, although it was sufficiently expressed even before hepatectomy. Suppression of p62/SQSTM1 increased FasL/Fas expression and reduced nuclear factor erythroid 2-related factor-2 (Nrf-2)-dependent antioxidant response elements activity and antioxidant responses in steatotic and nonsteatotic hepatocytes. Exogenously added FasL induced severe cellular OS and necrosis/apoptosis in steatotic hepatocytes, with only the necrosis being inhibited by pretreatment with antioxidants, suggesting that FasL/Fas-induced OS mainly leads to necrosis. Furthermore, p62/SQSTM1 re-expression in the steatotic liver markedly reduced liver injury and improved liver regeneration. INNOVATION: This study is the first which demonstrates that reduced expression of p62/SQSTM1 plays a crucial role in posthepatectomy acute injury and delayed regeneration of steatotic liver, mainly via redox-dependent mechanisms. CONCLUSION: In the steatotic liver, reduced expression of p62/SQSTM1 induced FasL/Fas overexpression and suppressed antioxidant genes, mainly through Nrf-2 inactivation, which, along with the hypo-responsiveness of Akt, caused posthepatectomy necrotic/apoptotic liver injury and delayed regeneration, both mainly via a redox-dependent mechanism.