Molecular characterization, spatio-temporal expression patterns of crtc2 gene and its immune roles in yellow catfish (Pelteobagrus fulvidraco).

cAMP response element binding (CREB) protein 2 (CRTC2) is a transcriptional coactivator of CREB and plays an important role in the immune system. Thus far, the physiological roles of Crtc2 in teleost are still poorly understood. In this study, the crtc2 gene was identified and characterized from yellow catfish (Pelteobagrus fulvidraco; therefore, the gene is termed as pfcrtc2), and its evolutionary and molecular characteristics as well as potential immunity-related roles were investigated. Our results showed that the open reading frame of pfcrtc2 was 2346 bp in length, encoding a protein with 781 amino acids. Gene structure analysis revealed its existence of 14 exons and 13 introns. A phylogenetic analysis proved that the tree of crtc2 was clustered into five groups, exhibiting a similar evolutionary topology with species evolution. Multiple protein sequences alignment demonstrated high conservation of the crtc2 in various vertebrates with similar structure. Syntenic and gene structural comparisons further established that crtc2 was highly conserved, implying its similar roles in diverse vertebrates. Tissue distribution pattern detected by quantitative real-time PCR showed that the pfcrtc2 gene was almost expressed in all detected tissues except for eyes, with the highest expression levels in the gonad, indicating that Crtc2 may play important roles in various tissues. In addition, pfcrtc2 was transcribed at all developmental stages in yellow catfish, showing the highest expression levels at 12 h after fertilization. Finally, the transcriptional profiles of crtc2 were significantly increased in yellow catfishes injected with Aeromonas hydrophila or Poly I:C, which shared a consistent change pattern with four immune-related genes including IL-17A, IL-10, MAPKp38, and NF-κBp65, suggesting pfCrtc2 may play critical roles in preventing both exogenous bacteria and virus invasion. In summary, our findings lay a solid foundation for further studies on the functions of pfcrtc2, and provide novel genetic loci for developing new strategies to control disease outbreak in teleost.

Ceramide enhanced the hepatic glucagon response through regulation of CREB activity.

BACKGROUND & AIMS: Hyperglycemia is associated with lipid disorders in patients with diabetes. Ceramides are metabolites involved in sphingolipid metabolism that accumulate during lipid disorders and exert deleterious effects on glucose and lipid metabolism. However, the effects of ceramide on glucagon-mediated hepatic gluconeogenesis remain largely unknown. This study was designed to investigate the impact of ceramides on gluconeogenesis in the context of the hepatic glucagon response, with the aim of finding new pharmacological interventions for hyperglycemia in diabetes. METHODS: Liquid chromatography-mass spectrometry was used to quantify ceramide content in the serum of patients with diabetes. Primary hepatocytes were isolated from male C57BL/6J mice to study the effects of ceramide on hepatic glucose production. Immunofluorescence staining was performed to view cAMP-responsive element-binding protein (CREB)- regulated transcription co-activator 2 (CRTC2) nuclear translocation in hepatocytes. Serine palmitoyl-transferase, long chain base subunit 2 (Sptlc2) knockdown mice were generated using an adeno-associated virus containing shRNA, and hepatic glucose production was assessed glucagon tolerance and pyruvate tolerance tests in mice fed a normal chow diet and high-fat diet. RESULTS: Increased ceramide levels were observed in the serum of patients newly diagnosed with type 2 diabetes. De novo ceramide synthesis was activated in mice with metabolic disorders. Ceramide enhanced hepatic glucose production in primary hepatocytes. In contrast, genetic silencing of Sptlc2 prevented this process. Mechanistically, ceramides de-phosphorylate CRTC2 (Ser 171) and facilitate its translocation into the nucleus for CREB activation, thereby augmenting the hepatic glucagon response. Hepatic Sptlc2 silencing blocked ceramide generation in the liver and thus restrained the hepatic glucagon response in mice fed a normal chow diet and high-fat diet. CONCLUSIONS: These data indicate that ceramide serves as an intracellular messenger that augments hepatic glucose production by regulating CRTC2/CREB activity in the context of the hepatic glucagon response, suggesting that CRTC2 phosphorylation might be a potential node for pharmacological interventions to restrain the hyperglycemic response during fasting in diabetes.

Proline hydroxylation of CREB-regulated transcriptional coactivator 2 controls hepatic glucose metabolism.

Prolyl hydroxylase domain (PHD) enzymes change HIF activity according to oxygen signal; whether it is regulated by other physiological conditions remains largely unknown. Here, we report that PHD3 is induced by fasting and regulates hepatic gluconeogenesis through interaction and hydroxylation of CRTC2. Pro129 and Pro615 hydroxylation of CRTC2 following PHD3 activation is necessary for its association with cAMP-response element binding protein (CREB) and nuclear translocation, and enhanced binding to promoters of gluconeogenic genes by fasting or forskolin. CRTC2 hydroxylation-stimulated gluconeogenic gene expression is independent of SIK-mediated phosphorylation of CRTC2. Liver-specific knockout of PHD3 (PHD3 LKO) or prolyl hydroxylase-deficient knockin mice (PHD3 KI) show attenuated fasting gluconeogenic genes, glycemia, and hepatic capacity to produce glucose during fasting or fed with high-fat, high-sucrose diet. Importantly, Pro615 hydroxylation of CRTC2 by PHD3 is increased in livers of fasted mice, diet-induced insulin resistance or genetically obese ob/ob mice, and humans with diabetes. These findings increase our understanding of molecular mechanisms linking protein hydroxylation to gluconeogenesis and may offer therapeutic potential for treating excessive gluconeogenesis, hyperglycemia, and type 2 diabetes.

LKB1 controls inflammatory potential through CRTC2-dependent histone acetylation.

Deregulated inflammation is a critical feature driving the progression of tumors harboring mutations in the liver kinase B1 (LKB1), yet the mechanisms linking LKB1 mutations to deregulated inflammation remain undefined. Here, we identify deregulated signaling by CREB-regulated transcription coactivator 2 (CRTC2) as an epigenetic driver of inflammatory potential downstream of LKB1 loss. We demonstrate that LKB1 mutations sensitize both transformed and non-transformed cells to diverse inflammatory stimuli, promoting heightened cytokine and chemokine production. LKB1 loss triggers elevated CRTC2-CREB signaling downstream of the salt-inducible kinases (SIKs), increasing inflammatory gene expression in LKB1-deficient cells. Mechanistically, CRTC2 cooperates with the histone acetyltransferases CBP/p300 to deposit histone acetylation marks associated with active transcription (i.e., H3K27ac) at inflammatory gene loci, promoting cytokine expression. Together, our data reveal a previously undefined anti-inflammatory program, regulated by LKB1 and reinforced through CRTC2-dependent histone modification signaling, that links metabolic and epigenetic states to cell-intrinsic inflammatory potential.

CRTC2 promotes paclitaxel resistance by inducing autophagy in ovarian cancer in part via the PI3K-AKT signaling axis.

Background: Ovarian cancer is the most malignant gynecological disease, which seriously threatens female physical and mental health. Paclitaxel is a first-line chemotherapy drug in the clinical treatment of ovarian cancer, but drug resistance has become an important factor affecting the survival of ovarian cancer patients. However, the main mechanism of chemotherapy resistance in ovarian cancer remains unclear. In this study, we analyzed the Integrated Gene Expression Database (GEO) dataset using comprehensive bioinformatics tools to provide new therapeutic strategies and search for prognostic targets for ovarian cancer. Methods: Ovarian cancer related genes were extracted from GSE18520 by bioinformatics method. Differentially expressed genes (DEGs) were obtained by differential analysis, and related genes and functions were elucidated. The key gene CRTC2 was identified by prognostic analysis. Immunohistochemistry was used to detect the expression of CRTC2 in chemotherapy-resistant and chemotherapy-sensitive ovarian cancer tissues. Functional analysis (cell assay) confirmed the role of CRTC2 in paclitaxel resistance. Autophagy related proteins were detected by Western blot. Autophagy flux analysis was performed using the GFP/RFP-LC3 adenovirus reporter. Results: A total of 3,852 DEGs were identified in the GEO microarray dataset. Key genes were screened by prognostic analysis. We found that CRTC2 was highly expressed in chemoresistant tissues of ovarian cancer. In 110 patients with ovarian cancer, high expression of CRTC2 was associated with poorer prognostic factors and shorter survival. At the same time, we found that CRTC2 can promote the proliferation and invasion ability of ovarian cancer cells. In addition, CRTC2 can affect the expression of PI3K, AKT, autophagic flux and sensitivity to paclitaxel chemotherapy in ovarian cancer. Conclusion: CRTC2 can affect autophagy partially through PI3K-AKT signaling pathway, and then affect the sensitivity of ovarian cancer to paclitaxel chemotherapy. CRTC2 may be a potential predictor or target for ovarian cancer therapy.

Biological functions of CRTC2 and its role in metabolism-related diseases.

CREB-regulated transcription coactivator2 (CRTC2 or TORC2) is a transcriptional coactivator of CREB(cAMP response element binding protein), which affects human energy metabolism through cyclic adenosine phosphate pathway, Mammalian target of rapamycin (mTOR) pathway, Sterol regulatory element binding protein 1(SREBP1), Sterol regulatory element binding protein 2 (SREBP2) and other substances Current studies on CRTC2 mainly focus on glucose and lipid metabolism, relevant studies show that CRTC2 can participate in the occurrence and development of related diseases by affecting metabolic homeostasis. It has been found that Crtc2 acts as a signaling regulator for cAMP and Ca2 + signaling pathways in many cell types, and phosphorylation at ser171 and ser275 can regulate downstream biological functions by controlling CRTC2 shuttling between cytoplasm and nucleus.

The SIK1/CRTC2/CREB1 and TWIST1/PI3K/Akt/GSK3ß signaling pathways mediated by microRNA-25-3p are altered in the schizophrenic rat brain.

Schizophrenia is a group of severe mental disorders. MiR-25-3p was shown to be involved in various neuropsychiatric diseases and can regulate SIK1 and TWIST1. The CRTC2/CREB1 and PI3K/Akt/GSK3ß signaling pathways are downstream pathways of SIK1 and TWIST1, respectively. This study investigated whether miR-25-3p-mediated SIK1/CRTC2/CREB1 and TWIST1/PI3K/Akt/GSK3ß signaling pathways are present in an animal model relevant to schizophrenia. A schizophrenic rat model was established by using sub-chronic MK-801 administration. An RNA-seq test was performed to examine the differentially expressed genes (DEGs) in the rat prefrontal cortex (PFC). The mRNA levels of miR-25-3p, SIK1, and TWIST in the PFC and caudate putamen (CPu) were assessed by qRT-PCR. Phosphorylation of the SIK1/CRTC2/CREB1 and TWIST1/PI3K/Akt/GSK3ß pathways in the two brain regions was examined by Western blots. The RNA-seq data revealed down-regulated miR-25-3p expression and up-regulated SIK1 and TWIST1 mRNA expression induced by MK-801. Additionally, SIK1 and TWIST1 were shown to be possible downstream responders of miR-25-3p in previous studies. qRT-PCR confirmed the changes of miR-25-3p, SIK1, and TWIST1 induced by MK-801 in both brain regions, which, however, was reversed by risperidone. Furthermore, the phosphorylation of the SIK1/CRTC2/CREB1 pathway was repressed by MK-801, whereas the phosphorylation of the TWIST1/PI3K/Akt/GSK3ß pathway was increased by MK-801 in either of the two brain regions. Moreover, the altered phosphorylation of these two signaling pathways induced by MK-801 can be restored by risperidone. In conclusion, this study suggests that altered SIK1/CRTC2/CREB1 and TWIST1/PI3K/Akt/GSK3ß signaling pathways mediated by miR-25-3p is very likely to be associated with schizophrenia, revealing potential targets for the treatment and clinical diagnosis of schizophrenia.

Preoperative administration of branched-chain amino acids reduces postoperative insulin resistance in rats by reducing liver gluconeogenesis.

BACKGROUND: Postoperative insulin resistance (PIR) represents an important characteristic of metabolic response following surgical injury. Clinical outcomes are negatively correlated to postoperative insulin resistance and hyperglycemia, indicating a novel treatment for reducing postoperative insulin resistance is urgently needed. The current work aimed to assess the protective effects of branched-chain amino acids (BCAA) on glucose metabolism disorders induced surgically in a rat model, and to explore the underpinning mechanism. METHODS AND RESULTS: Rats were randomly assigned to 2 groups, including the control and BCAA groups. Rats were given a compulsory oral 3 mL load by gavage two hours before surgery. The results showed that BCAA remarkably reduced glycemia by suppressing liver gluconeogenesis via reduction of cAMP-response element-binding protein-regulated transcription coactivator 2 (CRTC2) and glucose-6-phosphatase (G6PC) gene and protein expression levels (all Ps < 0.05). CONCLUSIONS: This study revealed that BCAA lower blood glucose levels by reducing liver gluconeogenesis without significant elevation of plasma insulin levels. We anticipate that preoperative BCAA supplementation may be a means for preventing postoperative insulin resistance.

Hepatic Sam68 Regulates Systemic Glucose Homeostasis and Insulin Sensitivity.

Hepatic glucose production (HGP) is an important component of glucose homeostasis, and deregulated HGP, particularly through gluconeogenesis, contributes to hyperglycemia and pathology of type-2 diabetes (T2D). It has been shown that the gluconeogenic gene expression is governed primarily by the transcription factor cAMP-response element (CRE)-binding protein (CREB) and its coactivator, CREB-regulated transcriptional coactivator 2 (CRTC2). Recently, we have discovered that Sam68, an adaptor protein and Src kinase substrate, potently promotes hepatic gluconeogenesis by promoting CRTC2 stability; however, the detailed mechanisms remain unclear. Here we show that in response to glucagon, Sam68 increases CREB/CRTC2 transactivity by interacting with CRTC2 in the CREB/CRTC2 complex and occupying the CRE motif of promoters, leading to gluconeogenic gene expression and glucose production. In hepatocytes, glucagon promotes Sam68 nuclear import, whereas insulin elicits its nuclear export. Furthermore, ablation of Sam68 in hepatocytes protects mice from high-fat diet (HFD)-induced hyperglycemia and significantly increased hepatic and peripheral insulin sensitivities. Thus, hepatic Sam68 potentiates CREB/CRTC2-mediated glucose production, contributes to the pathogenesis of insulin resistance, and may serve as a therapeutic target for T2D.

Mechanism of negative modulation of FSH signaling by salt-inducible kinases in rat granulosa cells.

The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). Recent findings revealed that salt-inducible kinases (SIKs) inhibit FSH actions in humans and rodents. This report seeks to increase our understanding of the molecular mechanisms controlled by SIKs that participate in the inhibition of FSH actions in primary rat granulosa cells (GCs). The results showed that FSH causes a transient induction of Sik1 mRNA. In contrast, SIK inhibition had no effects on FSH receptor expression. Next, we determined whether SIK inhibition enhances the effect of several sequential direct activators of the FSH signaling pathway. The findings revealed that SIK inhibition stimulates the induction of steroidogenic genes by forskolin, cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB). Strikingly, FSH stimulation of CREB and AKT phosphorylation was not affected by SIK inhibition. Therefore, we analyzed the expression and activation of putative CREB cofactors and demonstrated that GCs express CREB-regulated transcriptional coactivators (CRTC2) and that FSH treatment and SIK inhibition increase the nuclear expression of this factor. We concluded that SIKs target the FSH pathway by affecting factors located between cAMP/PKA and CREB and propose that SIKs control the activity of CRTC2 in ovarian GCs. The findings demonstrate for the first time that SIKs blunt the response of GCs to FSH, cAMP, PKA, and CREB, providing further evidence for a crucial role for SIKs in regulating ovarian function and female fertility.

Gestational bisphenol A exposure impairs hepatic lipid metabolism by altering mTOR/CRTC2/SREBP1 in male rat offspring.

Lipid metabolism is an important biochemical process in the body. Recent studies have found that environmental endocrine disruptors play an important role in the regulation of lipid metabolism. Bisphenol A (BPA), a common environmental endocrine disruptor, has adverse effects on lipid metabolism, but the mechanism is still unclear. This study aimed to investigate the effects of gestational BPA exposure on hepatic lipid metabolism and its possible mechanism in male offspring. The pregnant Sprague-Dawley rats were exposed to BPA (0, 0.05, 0.5, 5 mg/kg/day) from day 5 to day 19 of gestation to investigate the levels of triglyceride (TG) and total cholesterol (TC), and the expression of liver lipid metabolism-related genes in male offspring rats. The results showed that compared with the control group, the TG and TC levels in serum and liver in BPA-exposed groups was increased. And the expressions of liver fatty acid oxidation related genes, such as peroxisome proliferators-activated receptor α (PPARα) and carnitine palmitoyl transferase 1α (CPT1α), were down-regulated. However, the expressions of fatty acid synthesis related genes, such as sterol regulatory element binding proteins 1 (SREBP-1), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD-1), were up-regulated. The increased protein levels of mTOR and p-CRTC2 suggested that CREB-regulated transcription coactivator 2 (CRTC2) might be an important mediator in the mTOR/SREBP-1 pathway. In conclusion, these results demonstrated that mTOR/CRTC2/SREBP-1 could be affected by gestational BPA exposure, which may involve in the lipid metabolic disorders in later life.

CircSETBP1 Acts as a MiR-149-5p Sponge to Promote Intramuscular Fat Deposition by Regulating CRTCs.

Circular RNAs (circRNAs) appear to be crucial in the process of adipogenesis according to mounting data. CircSETBP1 is a newly discovered circRNA associated with adipogenesis. Sequencing verification and RNase R treatment have confirmed the circular nature of circSETBP1 in porcine tissue. The precise function and mechanism of circSETBP1 in adipocyte biology are still unclear. Cell counting kit-8 (CCK8), Oil red O staining, and quantitative real-time polymerase chain reaction (qRT-PCR) were employed in this investigation to reveal the functions of circSETBP1 and miR-149-5p in the growth and development of porcine intramuscular (IM) preadipocytes. CircSETBP1 overexpression accelerated cell differentiation while reducing cell proliferation. The opposite outcome was produced by overexpressing miR-149-5p. Meanwhile, circSETBP1 down-regulated the expression of miR-149-5p and miR-149-5p restrained the expression of CRTC1/CRTC2. CircSETBP1 was directly targeted by miR-149-5p, and CRTC1/CRTC2 were the target genes of miR-149-5p using bioinformatic analysis, the dual-Luciferase reporter system, and qRT-PCR. In conclusion, circSETBP1 controls the proliferation and differentiation of porcine IM preadipocytes and 3T3-L1 cells by regulating the miR-149-5p/CRTCs axis. The results of this study not only illuminate the molecular mechanism of circSETBP1/miR-149-5p involved in the deposition of porcine intramuscular fat (IMF), but they also provide a significant theoretical reference for raising quality of pork.

Salt-inducible kinase 2 (SIK2) inhibitor ARN-3236 attenuates bleomycin-induced pulmonary fibrosis in mice.

BACKGROUND: Pulmonary fibrosis is a fatal lung disease with complex pathogenesis and limited effective therapies. Salt-inducible kinase 2 (SIK2) is a kinase that phosphorylates CRTCs and regulates many physiological processes. However, the role of SIK2 on pulmonary fibrosis remains unclear, and whether SIK2 inhibitor can attenuate pulmonary fibrosis is unknown. METHOD: We subjected human fetal lung fibroblasts (HFLs) to transforming growth factor-ß1 (5 ng/mL) for 12 h, and examined the expression of SIK2, CRTCs and pCRTCs in fibroblasts by western-blot. To address the roles of SIK2 and CRTCs involved in the progression of pulmonary fibrosis, HFLs were treated with a small-molecule inhibitor ARN-3236 or by siRNA-mediated knockdown of SIK2 expression. Pulmonary fibrosis model was established with mice by exposing to bleomycin, and assessed by H&E and Masson's trichrome staining. COL1A and α-SMA distributions were detected in lung tissues by immunohistochemical staining. RESULTS: We discovered that SIK2 and phosphorylated-CRTC2 were expressed at a low basal level in normal lung tissues and quiescent fibroblasts, but increased in fibrotic lung tissues and activated fibroblasts. Inhibition of SIK2 by ARN-3236 prevented the fibroblasts differentiation and extracellular matrix expression in HFLs and attenuated bleomycin-induced pulmonary fibrosis in mice. Mechanistically, inactivation of SIK2 resulted in the dephosphorylation and nuclear translocation of CRTC2. Within the nucleus, CRTC2 binds to CREB, promoting CREB-dependent anti-fibrotic actions. CONCLUSION: In conclusion, our results elucidated a previously unexplored role of SIK2 in pulmonary fibrosis, and identified SIK2 as a new target for anti-fibrosis medicines.

Endoplasmic reticulum stress downregulates PGC-1α in skeletal muscle through ATF4 and an mTOR-mediated reduction of CRTC2.

BACKGROUND: Peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) downregulation in skeletal muscle contributes to insulin resistance and type 2 diabetes mellitus. Here, we examined the effects of endoplasmic reticulum (ER) stress on PGC-1α levels in muscle and the potential mechanisms involved. METHODS: The human skeletal muscle cell line LHCN-M2 and mice exposed to different inducers of ER stress were used. RESULTS: Palmitate- or tunicamycin-induced ER stress resulted in PGC-1α downregulation and enhanced expression of activating transcription factor 4 (ATF4) in human myotubes and mouse skeletal muscle. Overexpression of ATF4 decreased basal PCG-1α expression, whereas ATF4 knockdown abrogated the reduction of PCG-1α caused by tunicamycin in myotubes. ER stress induction also activated mammalian target of rapamycin (mTOR) in myotubes and reduced the nuclear levels of cAMP response element-binding protein (CREB)-regulated transcription co-activator 2 (CRTC2), a positive modulator of PGC-1α transcription. The mTOR inhibitor torin 1 restored PCG-1α and CRTC2 protein levels. Moreover, siRNA against S6 kinase, an mTORC1 downstream target, prevented the reduction in the expression of CRTC2 and PGC-1α caused by the ER stressor tunicamycin. CONCLUSIONS: Collectively, these findings demonstrate that ATF4 and the mTOR-CRTC2 axis regulates PGC-1α transcription under ER stress conditions in skeletal muscle, suggesting that its inhibition might be a therapeutic target for insulin resistant states. Video Abstract.

cAMP-Induced Nuclear Condensation of CRTC2 Promotes Transcription Elongation and Cystogenesis in Autosomal Dominant Polycystic Kidney Disease.

Formation of biomolecular condensates by phase separation has recently emerged as a new principle for regulating gene expression in response to extracellular signaling. However, the molecular mechanisms underlying the coupling of signal transduction and gene activation through condensate formation, and how dysregulation of these mechanisms contributes to disease progression, remain elusive. Here, the authors report that CREB-regulated transcription coactivator 2 (CRTC2) translocates to the nucleus and forms phase-separated condensates upon activation of cAMP signaling. They show that intranuclear CRTC2 interacts with positive transcription elongation factor b (P-TEFb) and activates P-TEFb by disrupting the inhibitory 7SK snRNP complex. Aberrantly elevated cAMP signaling plays central roles in the development of autosomal dominant polycystic kidney disease (ADPKD). They find that CRTC2 localizes to the nucleus and forms condensates in cystic epithelial cells of both mouse and human ADPKD kidneys. Genetic depletion of CRTC2 suppresses cyst growth in an orthologous ADPKD mouse model. Using integrative transcriptomic and cistromic analyses, they identify CRTC2-regulated cystogenesis-associated genes, whose activation depends on CRTC2 condensate-facilitated P-TEFb recruitment and the release of paused RNA polymerase II. Together, their findings elucidate a mechanism by which CRTC2 nuclear condensation conveys cAMP signaling to transcription elongation activation and thereby promotes cystogenesis in ADPKD.

Ilexgenin A restrains CRTC2 in the cytoplasm to prevent SREBP1 maturation via AMP kinase activation in the liver.

BACKGROUND AND PURPOSE: Ilexgenin A is a triterpenoid from ShanLv Cha with beneficial effects on metabolic homeostasis. We investigated whether ilexgenin A could inhibit hepatic de novo fatty acid synthesis via the interfering with SREBP1 maturation. EXPERIMENTAL APPROACH: The effects of Ilexgenin A on CRTC2 translocation and SREBP1 maturation were investigated in the liver of fasted mice and hepatocytes exposed to saturated fatty acids. The effect of Iilexgenin A on hepatic lipid accumulation was also observed in high-fat diet fed mice. KEY RESULTS: Sec23A and Sec31A are two subunits of COPII complex and their interaction is essential for the processing of SREBP1 maturation. Ilexgenin A activates AMPK by reducing cellular energy and preventing cytoplasmic CRTC2 to compete with Sec23A for binding to Sec31A under nutrient-rich conditions. Consequently, ilexgenin A impaired COPII-dependent SREBP1 maturation via disrupting Sec31A-Sec23A interaction, leading to the inhibition of de novo fatty acid synthesis in the liver. In contrast, mTORC1 phosphorylated Ser136 of CRTC2, facilitating the formation of Sec31A-Sec23A interaction to promote SREBP1 maturation, whereas this action was reversed by ilexgenin A in an AMPK-dependent manner. Ilexgenin A protected CRTC2 function and restrained hepatic lipogenic response in high fat diet-fed mice, providing in vivo evidence to support the beneficial effects of ilexgenin A on lipid metabolism. CONCLUSIONS AND IMPLICATIONS: Ilexgenin A activated AMPK and restrained CRTC2 to the cytoplasm to prevent SREBP1 maturation via impairing COPII function in the liver. This suggests that CRTC2 might be a potential target for pharmacological intervention to prevent hepatic lipid deposition. LINKED ARTICLES: This article is part of a themed issue on Preclinical Models for Cardiovascular disease research (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.5/issuetoc.

A novel role of CRTC2 in promoting nonalcoholic fatty liver disease.

OBJECTIVE: Diet-induced obesity is often associated with nonalcoholic fatty liver disease (NAFLD), which instigates severe metabolic disorders, including cirrhosis, hepatocellular carcinoma, and type 2 diabetes. We have shown that hepatic depletion of CREB regulated transcription co-activator (CRTC) 2 protects mice from the progression of diet-induced fatty liver phenotype, although the exact mechanism by which CRTC2 modulates this process is elusive to date. Here, we investigated the role of hepatic CRTC2 in the instigation of NAFLD in mammals. METHODS: Crtc2 liver-specific knockout (Crtc2 LKO) mice and Crtc2 flox/flox (Crtc2 f/f) mice were fed a high fat diet (HFD) for 7-8 weeks. Body weight, liver weight, hepatic lipid contents, and plasma triacylglycerol (TG) levels were determined. Western blot analysis was performed to determine Sirtuin (SIRT) 1, tuberous sclerosis complex (TSC) 2, and mammalian target of rapamycin complex (mTORC) 1 activity in the liver. Effects of Crtc2 depletion on lipogenesis was determined by measuring lipogenic gene expression (western blot analysis and qRT-PCR) in the liver as well as Oil red O staining in hepatocytes. Effects of miR-34a on mTORC1 activity and hepatic lipid accumulation was assessed by AAV-miR-34a virus in mice and Ad-miR-34a virus and Ad-anti-miR-34a virus in hepatocytes. Autophagic flux was assessed by western blot analysis after leupeptin injection in mice and bafilomycin treatment in hepatocytes. Lipophagy was assessed by transmission electron microscopy and confocal microscopy. Expression of CRTC2 and p-S6K1 in livers of human NAFLD patients was assessed by immunohistochemistry. RESULTS: We found that expression of CRTC2 in the liver is highly induced upon HFD-feeding in mice. Hepatic depletion of Crtc2 ameliorated HFD-induced fatty liver disease phenotypes, with a pronounced inhibition of the mTORC1 pathway in the liver. Mechanistically, we found that expression of TSC2, a potent mTORC1 inhibitor, was enhanced in Crtc2 LKO mice due to the decreased expression of miR-34a and the subsequent increase in SIRT1-mediated deacetylation processes. We showed that ectopic expression of miR-34a led to the induction of mTORC1 pathway, leading to the hepatic lipid accumulation in part by limiting lipophagy and enhanced lipogenesis. Finally, we found a strong association of CRTC2, miR-34a and mTORC1 activity in the NAFLD patients in humans, demonstrating a conservation of signaling pathways among species. CONCLUSIONS: These data collectively suggest that diet-induced activation of CRTC2 instigates the progression of NAFLD by activating miR-34a-mediated lipid accumulation in the liver via the simultaneous induction of lipogenesis and inhibition of lipid catabolism. Therapeutic approach to specifically inhibit CRTC2 activity in the liver could be beneficial in combating NAFLD in the future.

Activation of Crtc2/Creb1 in skeletal muscle enhances weight loss during intermittent fasting.

The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, maintenance of higher energy expenditure during the fast and reduced binge-eating during the feeding period. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

Molecular Brightness Approach for FRET Analysis of Donor-Linker-Acceptor Constructs at the Single Molecule Level: A Concept.

In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1-linker-mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery.

A kinome screen reveals that Nemo-like kinase is a key suppressor of hepatic gluconeogenesis.

Antihyperglycemic therapy is an important priority for the treatment of type 2 diabetes (T2D). Excessive hepatic glucose production (HGP) is a major cause of fasting hyperglycemia. Therefore, a better understanding of its regulation would be important to develop effective antihyperglycemic therapies. Using a gluconeogenesis-targeted kinome screening approach combined with transcriptome analyses, we uncovered Nemo-like kinase (NLK) as a potent suppressor of HGP. Mechanistically, NLK phosphorylates and promotes nuclear export of CRTC2 and FOXO1, two key regulators of hepatic gluconeogenesis, resulting in the proteasome-dependent degradation of the former and the inhibition of the self-transcriptional activity and expression of the latter. Importantly, the expression of NLK is downregulated in the liver of individuals with diabetes and in diabetic rodent models and restoring NLK expression in the mouse model ameliorates hyperglycemia. Therefore, our findings uncover NLK as a critical player in the gluconeogenic regulatory network and as a potential therapeutic target for T2D.