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.

Deficiency of the Mitochondrial NAD Kinase Causes Stress-Induced Hepatic Steatosis in Mice.

BACKGROUND & AIMS: The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (NADK2, also called MNADK) catalyzes phosphorylation of NAD to yield NADP. Little is known about the functions of mitochondrial NADP and MNADK in liver physiology and pathology. We investigated the effects of reduced mitochondrial NADP by deleting MNADK in mice. METHODS: We generated MNADK knockout (KO) mice on a C57BL/6NTac background; mice with a wild-type Mnadk gene were used as controls. Some mice were placed on an atherogenic high-fat diet (16% fat, 41% carbohydrate, and 1.25% cholesterol supplemented with 0.5% sodium cholate) or given methotrexate intraperitoneally. We measured rates of fatty acid oxidation in primary hepatocytes using radiolabeled palmitate and in mice using indirect calorimetry. We measured levels of reactive oxygen species in mouse livers and primary hepatocytes. Metabolomic analyses were used to quantify serum metabolites, such as amino acids and acylcarnitines. RESULTS: The KO mice had metabolic features of MNADK-deficient patients, such as increased serum concentrations of lysine and C10:2 carnitine. When placed on the atherogenic high-fat diet, the KO mice developed features of nonalcoholic fatty liver disease and had increased levels of reactive oxygen species in livers and primary hepatocytes, compared with control mice. During fasting, the KO mice had a defect in fatty acid oxidation. MNADK deficiency reduced the activation of cAMP-responsive element binding protein-hepatocyte specific and peroxisome proliferator-activated receptor alpha, which are transcriptional activators that mediate the fasting response. The activity of mitochondrial sirtuins was reduced in livers of the KO mice. Methotrexate inhibited the catalytic activity of MNADK in hepatocytes and in livers in mice with methotrexate injection. In mice given injections of methotrexate, supplementation of a diet with nicotinamide riboside, an NAD precursor, replenished hepatic NADP and protected the mice from hepatotoxicity, based on markers such as increased level of serum alanine aminotransferase. CONCLUSION: MNADK facilitates fatty acid oxidation, counteracts oxidative damage, maintains mitochondrial sirtuin activity, and prevents metabolic stress-induced non-alcoholic fatty liver disease in mice.

Toll-like Receptor (TLR) Signaling Interacts with CREBH to Modulate High-density Lipoprotein (HDL) in Response to Bacterial Endotoxin.

Bacterial endotoxin can induce inflammatory and metabolic changes in the host. In this study, we revealed a molecular mechanism by which a stress-inducible, liver-enriched transcription factor, cAMP-responsive element-binding protein hepatic-specific (CREBH), modulates lipid profiles to protect the liver from injuries upon the bacterial endotoxin lipopolysaccharide (LPS). LPS challenge can activate CREBH in mouse liver tissues in a toll-like receptor (TLR)/MyD88-dependent manner. Upon LPS challenge, CREBH interacts with TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase that functions as a key mediator of TLR signaling, and this interaction relies on MyD88. Further analysis demonstrated that TRAF6 mediates K63-linked ubiquitination of CREBH to facilitate CREBH cleavage and activation. CREBH directly activates expression of the gene encoding Apolipoprotein A4 (ApoA4) under LPS challenge, leading to modulation of high-density lipoprotein (HDL) in animals. CREBH deficiency led to reduced production of circulating HDL and increased liver damage upon high-dose LPS challenge. Therefore, TLR/MyD88-dependent, TRAF6-facilitated CREBH activation represents a mammalian hepatic defense response to bacterial endotoxin by modulating HDL.

Exposure to fine airborne particulate matters induces hepatic fibrosis in murine models.

BACKGROUND & AIMS: Hepatic fibrosis, featured by the accumulation of excessive extracellular matrix in liver tissue, is associated with metabolic disease and cancer. Inhalation exposure to airborne particulate matter in fine ranges (PM2.5) correlates with pulmonary dysfunction, cardiovascular disease, and metabolic syndrome. In this study, we investigated the effect and mechanism of PM2.5 exposure on hepatic fibrogenesis. METHODS: Both inhalation exposure of mice and in vitro exposure of specialized cells to PM2.5 were performed to elucidate the effect of PM2.5 exposure on hepatic fibrosis. Histological examinations, gene expression analyses, and genetic animal models were utilized to determine the effect and mechanism by which PM2.5 exposure promotes hepatic fibrosis. RESULTS: Inhalation exposure to concentrated ambient PM2.5 induces hepatic fibrosis in mice under the normal chow or high-fat diet. Mice after PM2.5 exposure displayed increased expression of collagens in liver tissues. Exposure to PM2.5 led to activation of the transforming growth factor ß-SMAD3 signaling, suppression of peroxisome proliferator-activated receptor γ, and expression of collagens in hepatic stellate cells. NADPH oxidase plays a critical role in PM2.5-induced liver fibrogenesis. CONCLUSIONS: Exposure to PM2.5 exerts discernible effects on promoting hepatic fibrogenesis. NADPH oxidase mediates the effects of PM2.5 exposure on promoting hepatic fibrosis.

MCP-1-induced protein attenuates post-infarct cardiac remodeling and dysfunction through mitigating NF-κB activation and suppressing inflammation-associated microRNA expression.

MCP-1-induced protein (MCPIP, also known as ZC3H12A) has recently been uncovered to act as a negative regulator of inflammation. Expression of MCPIP was elevated in the ventricular myocardium of patients with ischemic heart failure. However, the role of MCPIP in the development of post-infarct cardiac inflammation and remodeling is unknown. The objective of the present study was to investigate whether MCPIP exerts an inhibitory effect on the cardiac inflammatory response and adverse remodeling after myocardial infarction (MI). Mice with cardiomyocyte-specific expression of MCPIP and their wild-type littermates (FVB/N) were subjected to permanent ligation of left coronary artery. The levels of MCPIP were significantly increased in the ischemic myocardium and sustained for 4 weeks after MI. Acute infarct size was comparable between groups. However, constitutive overexpression of MCPIP in the murine heart resulted in improved survival rate, decreased cardiac hypertrophy, less of fibrosis and scar formation, and better cardiac performance at 28 days after MI, along with a markedly reduced monocytic cell infiltration, less cytokine expression, decreased caspase-3/7 activities and apoptotic cell death compared to the wild-type hearts. Cardiomyocyte-specific expression of MCPIP also attenuated activation of cardiac NF-κB signaling and expression of inflammation-associated microRNAs (miR-126, -146a, -155, and -199a) when compared with the post-infarct wild-type hearts. In vitro, MCPIP expression suppressed hypoxia-induced NF-κB-luciferase activity in cardiomyocytes. In conclusion, MCPIP expression in the ischemic myocardium protects against adverse cardiac remodeling and dysfunction following MI by modulation of local myocardial inflammation, possibly through mitigating NF-κB signaling and suppressing inflammation-associated microRNA expression.

Effects of Aerobic vs. Resistance Exercise on Vascular Function and Vascular Endothelial Growth Factor in Older Women.

This study aimed to investigate the effects of different types of exercise (aerobic vs. resistance) on vascular function and vascular endothelial growth factor in older women. Forty-three older women, aged 65-75 years old, voluntarily participated in this study. All participants were randomly assigned to one of the following three groups: aerobic exercise (AE; n = 14), resistance exercise (RE; n = 15), and control (CG; n = 14) groups. All participants in the exercise groups performed their respective exercises for 60 min/day, three days/week, for 16 weeks. The intensity of aerobic and resistance exercises was determined using the individual heart rate reserve (40-60%) and RPE (12-13), respectively. The vascular function test included the brachial-ankle pulse wave velocity (ba-PWV), carotid artery blood flow volume, and velocity. Participants' blood samples were collected to analyze the vascular endothelial growth factor (VEGF). A significance level of 0.05 was set. Our results showed that ba-PWV improved following both AE (14.5%) and RE groups (11.1%) (all p < 0.05). Increases in carotid blood flow volume (AE: 15.4%, RE: 18.6%) and total artery peak velocity (AE: 20.4%, RE: 17%) were observed in AE and RE groups (p < 0.05), while flow total artery mean velocity (36.2%) and peak velocities (20.5%) were only increased in the aerobic exercise group (p < 0.05). VEGF was increased after resistance exercise (p < 0.05). Overall, aerobic exercise provides greater benefits on vascular function than resistance exercise but further research is needed on VEGF regarding whether this change is associated with vascular function improvement in older women.

A hepatokine derived from the ER protein CREBH promotes triglyceride metabolism by stimulating lipoprotein lipase activity.

The endoplasmic reticulum (ER)-tethered, liver-enriched stress sensor CREBH is processed in response to increased energy demands or hepatic stress to release an amino-terminal fragment that functions as a transcription factor for hepatic genes encoding lipid and glucose metabolic factors. Here, we discovered that the carboxyl-terminal fragment of CREBH (CREBH-C) derived from membrane-bound, full-length CREBH was secreted as a hepatokine in response to fasting or hepatic stress. Phosphorylation of CREBH-C mediated by the kinase CaMKII was required for efficient secretion of CREBH-C through exocytosis. Lipoprotein lipase (LPL) mediates the lipolysis of circulating triglycerides for tissue uptake and is inhibited by a complex consisting of angiopoietin-like (ANGPTL) 3 and ANGPTL8. Secreted CREBH-C blocked the formation of ANGPTL3-ANGPTL8 complexes, leading to increased LPL activity in plasma and metabolic tissues in mice. CREBH-C administration promoted plasma triglyceride clearance and partitioning into peripheral tissues and mitigated hypertriglyceridemia and hepatic steatosis in mice fed a high-fat diet. Individuals with obesity had higher circulating amounts of CREBH-C than control individuals, and human CREBH loss-of-function variants were associated with dysregulated plasma triglycerides. These results identify a stress-induced, secreted protein fragment derived from CREBH that functions as a hepatokine to stimulate LPL activity and triglyceride homeostasis.

Boosting UPR transcriptional activator XBP1 accelerates acute wound healing.

Patients' suffering from large or deep wounds caused by traumatic and/or thermal injuries have significantly lower chances of recapitulating lost skin function through natural healing. We tested whether enhanced unfolded protein response (UPR) by expression of a UPR transcriptional activator, X-box-binding protein 1 (XBP1) can significantly promote wound repair through stimulating growth factor production and promoting angiogenesis. In mouse models of a second-degree thermal wound, a full-thickness traumatic wound, and a full-thickness diabetic wound, the topical gene transfer of the activated form of XBP1 (spliced XBP1, XBP1s) can significantly enhance re-epithelialization and increase angiogenesis, leading to rapid, nearly complete wound closure with intact regenerated epidermis and dermis. Overexpression of XBP1s stimulated the transcription of growth factors in fibroblasts critical to proliferation and remodeling during wound repair, including platelet-derived growth factor BB, basic fibroblast growth factor, and transforming growth factor beta 3. Meanwhile, the overexpression of XBP1s boosted the migration and tube formation of dermal microvascular endothelial cells in vitro. Our functional and mechanistic investigations of XBP1-mediated regulation of wound healing processes provide novel insights into the previously undermined physiological role of the UPR in skin injuries. The finding opens an avenue to developing potential XBP1-based therapeutic strategies in clinical wound care protocols.

Homocysteine-induced endoplasmic reticulum stress activates FGF21 and is associated with browning and atrophy of white adipose tissue in Bhmt knockout mice.

Betaine-homocysteine methyltransferase (BHMT) catalyzes the transfer of methyl groups from betaine to homocysteine (Hcy), producing methionine and dimethylglycine. In this work, we characterize Bhmt wild type (Bhmt-WT) and knockout (Bhmt-KO) mice that were fully backcrossed to a C57Bl6/J background. Consistent with our previous findings, Bhmt-KO mice had decreased body weight, fat mass, and adipose tissue weight compared to WT. Histological analyses and gene expression profiling indicate that adipose browning was activated in KO mice and contributed to the adipose atrophy observed. BHMT is not expressed in adipose tissue but is abundant in liver; thus, a signal must originate from the liver that modulates adipose tissue. We found that, in Bhmt-KO mice, homocysteine-induced endoplasmic reticulum (ER) stress is associated with activation of the hepatic transcription factor cyclic AMP response element binding protein (CREBH), and an increase in hepatic and plasma concentrations of fibroblast growth factor 21 (FGF21), which is known to induce adipose browning. Our data indicate that the deletion of a single gene in one-carbon metabolism modifies adipose biology and energy metabolism. Future studies could focus on identifying if functional polymorphisms in BHMT result in a similar adipose atrophy phenotype.

Analysis of Insulin Resistance in Nonalcoholic Steatohepatitis.

Insulin resistance is a major phenotype observed in nonalcoholic steatohepatitis (NASH), the advanced stage of nonalcoholic fatty liver disease (NAFLD). Insulin resistance in NASH is characterized by reductions in whole body, hepatic, and adipose tissue insulin sensitivity. The mechanisms underlying hepatic insulin resistance is primarily associated with hepatic glucose production (HGP) rate. Hepatic insulin resistance can also be a consequence or a driving factor of hepatic lipid accumulation by increasing free fatty acid synthesis, delivery, and catabolism. The common method to assess hepatic insulin resistance is to measure hepatic glucose production (HGP) using isotope tracer distribution technique. However, non-radioactive approaches have been developed to assess hepatic insulin resistance in the context of NASH. In this chapter, we describe the methods to evaluate hepatic insulin resistance in animal models of NASH by examining insulin sensitivity and glucose tolerance as well as the key molecules in hepatic insulin signaling pathways.

The links of fine airborne particulate matter exposure to occurrence of cardiovascular and metabolic diseases in Michigan, USA.

Air pollutants, particularly airborne particulate matter with aerodynamic diameter < 2.5µm (PM2.5), have been linked to the increase in mortality and morbidity associated with cardiovascular and metabolic diseases. In this study, we investigated the dose-risk relationships between PM2.5 concentrations and occurrences of cardiovascular and metabolic diseases as well as the confounding socioeconomic factors in Michigan, USA, where PM2.5 levels are generally considered acceptable. Multivariate linear regression analyses were performed to investigate the relationship between health outcome and annual ground-level PM2.5 concentrations of 82 counties in Michigan. The analyses revelated significant linear dose-response associations between PM2.5 concentrations and cardiovascular disease (CVD) hospitalization. A 10 µg/m3 increase in PM2.5 exposure was found to be associated with a 3.0% increase in total CVD, 0.45% increase in Stroke, and a 0.3% increase in Hypertension hospitalization rates in Medicare beneficiaries. While the hospitalization rates of Total Stroke, Hemorrhagic Stroke, and Hypertension in urbanized counties were significantly higher than those of rural counties, the death rates of coronary heart disease and ischemic stroke in urbanized counties were significantly lower than those of rural counties. These results were correlated with the facts that PM2.5 levels in urbanized counties were significantly higher than that in rural counties and that the percentage of the population with health insurance and the median household income in rural counties were significantly lower. While obesity prevalence showed evidence of a weak positive correlation (ρ = 0.20, p-value = 0.078) with PM2.5 levels, there was no significant dose-response association between county diabetes prevalence rates and PM2.5 exposure in Michigan. In summary, this study revealed strong dose-response associations between PM2.5 concentrations and CVD incidence in Michigan, USA. The socioeconomic factors, such as access to healthcare resources and median household income, represent important confounding factors that could override the impact of PM2.5 exposure on CVD mortality.

The mitochondrial NAD+ transporter SLC25A51 is a fasting-induced gene affecting SIRT3 functions.

INTRODUCTION: Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism and energy production. NAD+-dependent deacetylase sirtuin 3 (SIRT3) regulates the acetylation levels of mitochondrial proteins that are involved in mitochondrial homeostasis. Fasting up-regulates hepatic SIRT3 activity, which requires mitochondrial NAD+. What is the mechanism, then, to transport more NAD+ into mitochondria to sustain enhanced SIRT3 activity during fasting? OBJECTIVE: SLC25A51 is a recently discovered mitochondrial NAD+ transporter. We tested the hypothesis that, during fasting, increased expression of SLC25A51 is needed for enhanced mitochondrial NAD+ uptake to sustain SIRT3 activity. Because the fasting-fed cycle and circadian rhythm are closely linked, we further tested the hypothesis that SLC25A51 is a circadian regulated gene. METHODS: We examined Slc25a51 expression in the liver of fasted mice, and examined its circadian rhythm in wild-type mice and those with liver-specific deletion of the clock gene BMAL1 (LKO). We suppressed Slc25a51 expression in hepatocytes and the mouse liver using shRNA-mediated knockdown, and then examined mitochondrial NAD+ levels, SIRT3 activities, and acetylation levels of SIRT3 target proteins (IDH2 and ACADL). We measured mitochondrial oxygen consumption rate using Seahorse analysis in hepatocytes with reduced Slc25a51 expression. RESULTS: We found that fasting induced the hepatic expression of Slc25a51, and its expression showed a circadian rhythm-like pattern that was disrupted in LKO mice. Reduced expression of Slc25a51 in hepatocytes decreased mitochondrial NAD+ levels and SIRT3 activity, reflected by increased acetylation of SIRT3 targets. Slc25a51 knockdown reduced the oxygen consumption rate in intact hepatocytes. Mice with reduced Slc25a51 expression in the liver manifested reduced hepatic mitochondrial NAD+ levels, hepatic steatosis and hypertriglyceridemia. CONCLUSIONS: Slc25a51 is a fasting-induced gene that is needed for hepatic SIRT3 functions.

The mitochondrial NAD kinase functions as a major metabolic regulator upon increased energy demand.

OBJECTIVE: The mitochondrial nicotinamide adenine dinucleotide (NAD) kinase (MNADK) mediates de novo mitochondrial NADP biosynthesis by catalyzing the phosphorylation of NAD to yield NADP. In this study, we investigated the function and mechanistic basis by which MNADK regulates metabolic homeostasis. METHODS: Generalized gene set analysis by aggregating human patient genomic databases, metabolic studies with genetically engineered animal models, mitochondrial bioenergetic analysis, as well as gain- and loss- of-function studies were performed to address the functions and mechanistic basis by which MNADK regulates energy metabolism and redox state associated with metabolic disease. RESULTS: Human MNADK common gene variants or decreased expression of the gene are significantly associated with the occurrence of type-2 diabetes, non-alcoholic fatty liver disease (NAFLD), or hepatocellular carcinoma (HCC). Ablation of the MNADK gene in mice led to decreased fat oxidation, coincident with increased respiratory exchange ratio (RER) and decreased energy expenditure upon energy demand triggered by endurance exercise or fasting. On an atherogenic high-fat diet (HFD), MNADK-null mice exhibited hepatic insulin resistance and glucose intolerance, indicating a type-2 diabetes-like phenotype in the absence of MNADK. MNADK deficiency led to a decrease in mitochondrial NADP(H) but an increase in cellular reactive oxygen species (ROS) in mouse livers. Consistently, protein levels of the major metabolic regulators or enzymes were decreased, while their acetylation modifications were increased in the livers of MNADK-null mice. Feeding mice with a HFD caused S-nitrosylation (SNO) modification, a posttranslational modification that represses protein activities, on MNADK protein in the liver. Reconstitution of an SNO-resistant MNADK variant, MNADK-S193, into MNADK-null mice mitigated hepatic steatosis induced by HFD. CONCLUSION: MNADK, the only known mammalian mitochondrial NAD kinase, plays important roles in preserving energy homeostasis to mitigate the risk of metabolic disorders.

Taekwondo training and fitness in female adolescents.

In this study, we determined the specificity of a low frequency taekwondo training programme on physical fitness levels in adolescent females who receive limited physical education instruction (i.e. 2 days per week). Major components of physical fitness assessed were: skeletal muscle fitness (hand grip strength, bent arm hang, standing long jump, and isokinetic strength), flexibility (sit-and-reach test), speed and agility (10 × 5-m shuttle run), and cardiovascular fitness (VO(2max) and 20-m shuttle run). Changes in body composition were also assessed (dual X-ray absorptiometry, DXA). Participants were divided into two groups, a taekwondo training group (n = 21), which trained 50 min a day, 2 days per week for 12 weeks, and a control group (n = 10). Taekwondo training improved isokinetic strength, standing long jump, and sit-and-reach performance. Body fat mass and percent body fat were reduced. No changes in grip strength, bent arm hang time, speed and agility, or cardiorespiratory fitness were observed. Results indicate that low frequency taekwondo training in adolescent females produces beneficial changes in skeletal muscle fitness, flexibility, and body composition in a relatively short period of time. Consequently, this specific type of training can be useful to female adolescents in structured school environments where physical education classes are limited and there is little free time for physical activity.

Regulation of hepatic circadian metabolism by the E3 ubiquitin ligase HRD1-controlled CREBH/PPARα transcriptional program.

OBJECTIVE: The endoplasmic reticulum (ER)-resident E3 ligase HRD1 and its co-activator Sel1L are major components of ER-associated degradation (ERAD) machinery. Here, we investigated the molecular mechanism and functional significance underlying the circadian regulation of HRD1/Sel1L-mediated protein degradation program in hepatic energy metabolism. METHODS: Genetically engineered animal models as well as gain- and loss-of-function studies were employed to address the circadian regulatory mechanism and functional significance. Gene expression, transcriptional activation, protein-protein interaction, and animal metabolic phenotyping analyses were performed to dissect the molecular network and metabolic pathways. RESULTS: Hepatic HRD1 and Sel1L expression exhibits circadian rhythmicity that is controlled by the ER-tethered transcriptional activator CREBH, the nuclear receptor peroxisome proliferator-activated receptor α (PPARα), and the core clock oscillator BMAL1 in mouse livers. HRD1/Sel1L mediates polyubiquitination and degradation of the CREBH protein across the circadian cycle to modulate rhythmic expression of the genes encoding the rate-limiting enzymes or regulators in fatty acid (FA) oxidation, triglyceride (TG) lipolysis, lipophagy, and gluconeogenesis. HRD1 liver-specific knockout (LKO) mice displayed increased expression of the genes involved in lipid and glucose metabolism and impaired circadian profiles of circulating TG, FA, and glucose due to overproduction of CREBH. The circadian metabolic activities of HRD1 LKO mice were inversely correlated with those of CREBH KO mice. Suppressing CREBH overproduction in the livers of HRD1 LKO mice restored the diurnal levels of circulating TG and FA of HRD1 LKO mice. CONCLUSION: Our work revealed a key circadian-regulated molecular network through which the E3 ubiquitin ligase HRD1 and its co-activator Sel1L regulate hepatic circadian metabolism.

Type 2 diabetes sex-specific effects associated with E167K coding variant in TM6SF2.

The rs58542926C >T (E167K) variant of the transmembrane 6 superfamily member 2 gene (TM6SF2) is associated with increased risks for nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D). Nevertheless, the role of the TM6SF2 rs58542926 variant in glucose metabolism is poorly understood. We performed a sex-stratified analysis of the association between the rs58542926C >T variant and T2D in multiple cohorts. The E167K variant was significantly associated with T2D, especially in males. Using an E167K knockin (KI) mouse model, we found that male but not the female KI mice exhibited impaired glucose tolerance. As an ER membrane protein, TM6SF2 was found to interact with inositol-requiring enzyme 1 α (IRE1α), a primary ER stress sensor. The male Tm6sf2 KI mice exhibited impaired IRE1α signaling in the liver. In conclusion, the E167K variant of TM6SF2 is associated with glucose intolerance primarily in males, both in humans and mice.

The UPR Transducer IRE1 Promotes Breast Cancer Malignancy by Degrading Tumor Suppressor microRNAs.

Dysregulation of inositol-requiring enzyme 1 (IRE1), the primary transducer of Unfolded Protein Response (UPR), has been observed in tumor initiation and progression, but the underlying mechanism remains to be further elucidated. In this study, we identified that the IRE1 gene is frequently amplified and over-expressed in aggressive luminal B breast cancer cells and that IRE1 upregulation is significantly associated with worse overall survival of patients with breast cancer. IRE1 processes and mediates degradation of a subset of tumor suppressor microRNAs (miRNAs), including miR-3607, miR-374a, and miR-96, via a mechanism called Regulated IRE1-Dependent Decay (RIDD). IRE1-dependent degradation of tumor suppressor miR-3607 leads to elevation of RAS oncogene GTPase RAB3B in breast cancer cells. Inhibition of IRE1 endoribonuclease activity with the pharmacological compound 4µ8C or genetic approaches effectively suppresses luminal breast cancer cell proliferation and aggressive cancer phenotypes. Our work revealed the IRE1-RIDD-miRNAs pathway that promotes malignancy of luminal breast cancer.

IRE1α prevents hepatic steatosis by processing and promoting the degradation of select microRNAs.

Obesity or a high-fat diet represses the endoribonuclease activity of inositol-requiring enzyme 1α (IRE1α), a transducer of the unfolded protein response (UPR) in cells under endoplasmic reticulum (ER) stress. An impaired UPR is associated with hepatic steatosis and nonalcoholic fatty liver disease (NAFLD), which is caused by lipid accumulation in the liver. We found that IRE1α was critical to maintaining lipid homeostasis in the liver by repressing the biogenesis of microRNAs (miRNAs) that regulate lipid mobilization. In mice fed normal chow, the endoribonuclease function of IRE1α processed a subset of precursor miRNAs in the liver, including those of the miR-200 and miR-34 families, such that IRE1α promoted their degradation through the process of regulated IRE1-dependent decay (RIDD). A high-fat diet in mice or hepatic steatosis in patients was associated with the S-nitrosylation of IRE1α and inactivation of its endoribonuclease activity. This resulted in an increased abundance of these miRNA families in the liver and, consequently, a decreased abundance of their targets, which included peroxisome proliferator-activated receptor α (PPARα) and the deacetylase sirtuin 1 (SIRT1), regulators of fatty acid oxidation and triglyceride lipolysis. IRE1α deficiency exacerbated hepatic steatosis in mice. The abundance of the miR-200 and miR-34 families was also increased in cultured, lipid-overloaded hepatocytes and in the livers of patients with hepatic steatosis. Our findings reveal a mechanism by which IRE1α maintains lipid homeostasis through its regulation of miRNAs, a regulatory pathway distinct from the canonical IRE1α-UPR pathway under acute ER stress.

CREBH Maintains Circadian Glucose Homeostasis by Regulating Hepatic Glycogenolysis and Gluconeogenesis.

Cyclic AMP-responsive element binding protein, hepatocyte specific (CREBH), is a liver-enriched, endoplasmic reticulum-tethered transcription factor known to regulate the hepatic acute-phase response and lipid homeostasis. In this study, we demonstrate that CREBH functions as a circadian transcriptional regulator that plays major roles in maintaining glucose homeostasis. The proteolytic cleavage and posttranslational acetylation modification of CREBH are regulated by the circadian clock. Functionally, CREBH is required in order to maintain circadian homeostasis of hepatic glycogen storage and blood glucose levels. CREBH regulates the rhythmic expression of the genes encoding the rate-limiting enzymes for glycogenolysis and gluconeogenesis, including liver glycogen phosphorylase (PYGL), phosphoenolpyruvate carboxykinase 1 (PCK1), and the glucose-6-phosphatase catalytic subunit (G6PC). CREBH interacts with peroxisome proliferator-activated receptor α (PPARα) to synergize its transcriptional activities in hepatic gluconeogenesis. The acetylation of CREBH at lysine residue 294 controls CREBH-PPARα interaction and synergy in regulating hepatic glucose metabolism in mice. CREBH deficiency leads to reduced blood glucose levels but increases hepatic glycogen levels during the daytime or upon fasting. In summary, our studies revealed that CREBH functions as a key metabolic regulator that controls glucose homeostasis across the circadian cycle or under metabolic stress.

Interaction between stress responses and circadian metabolism in metabolic disease.

Circadian rhythms play crucial roles in orchestrating diverse physiological processes that are critical for health and disease. Dysregulated circadian rhythms are closely associated with various human metabolic diseases, including type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease. Modern lifestyles are frequently associated with an irregular circadian rhythm, which poses a significant risk to public health. While the central clock has a set periodicity, circadian oscillators in peripheral organs, particularly in the liver, can be entrained by metabolic alterations or stress cues. At the molecular level, the signal transduction pathways that mediate stress responses interact with, and are often integrated with, the key determinants of circadian oscillation, to maintain metabolic homeostasis under physiological or pathological conditions. In the liver, a number of nuclear receptors or transcriptional regulators, which are regulated by metabolites, hormones, the circadian clock, or environmental stressors, serve as direct links between stress responses and circadian metabolism. In this review, we summarize recent advances in the understanding of the interactions between stress responses (the endoplasmic reticulum (ER) stress response, the oxidative stress response, and the inflammatory response) and circadian metabolism, and the role of these interactions in the development of metabolic diseases.