RORα-GABP-TFAM axis alleviates myosteatosis with fatty atrophy through reinforcement of mitochondrial capacity.

BACKGROUND: Fat infiltration in muscle, called 'myosteatosis', precedes muscle atrophy, which subsequently results in sarcopenia. Myosteatosis is frequently observed in patients with nonalcoholic fatty liver disease (NAFLD). We have previously reported that retinoic acid receptor-related orphan receptor-α (RORα) regulates mitochondrial dynamics and mitophagy in hepatocytes, resulting in an alleviation of NAFLD. In this study, we aimed to investigate the role of RORα in skeletal muscle and to understand molecular mechanisms by which RORα controls mitochondrial capacity, using an NAFLD-associated myosteatosis mouse model. METHODS: To establish a myosteatosis model, 7-week-old C57BL/6N mice were fed with high-fat diet (HFD). After 15 weeks of diet feeding, an adeno-associated virus vector encoding RORα (AAV-RORα) was injected to gastrocnemius (GA) muscles, or after 7 weeks of HFD feeding, JC1-40, an RORα agonistic ligand, was administered daily at a dose of 5 mg/kg/day by oral gavage for 5 weeks. Histological, biochemical and molecular analyses in various in vivo and in vitro experiments were performed. RESULTS: First, the number of oxidative MyHC2a fibres with intensive lipid infiltration increased by 3.8-fold in the red region of the GA of mice with myosteatosis (P < 0.001). RORα was expressed around MyHC2a fibres, and its level increased by 2.7-fold after HFD feeding (P < 0.01). Second, treatment of RORα ligands in C2C12 myoblasts, such as cholesterol sulfate and JC1-40, enhanced the number of oxidative fibres stained for MyHC1 and MyHC2a by two-fold to four-fold (P < 0.01), while it reduced the lipid levels in MyHC2a fibres by 20-50% (P < 0.001) in the presence of palmitic acids. Third, mitochondrial membrane potential (P < 0.01) and total area of mitochondria (P < 0.01) were enhanced by treatment of these ligands. Chromatin immunoprecipitation analysis showed that RORα bound the promoter of GA-binding protein α subunit gene that led to activation of mitochondrial transcription factor A (TFAM) in C2C12 myoblasts (P < 0.05). Finally, intramuscular transduction of AAV-RORα alleviated the HFD-induced myosteatosis with fatty atrophy; lipid contents in MyHC2a fibres decreased by 48% (P < 0.001), whereas the number of MyHC2b fibre increased by 22% (P < 0.001). Also, administration of JC1-40 improved the signs of myosteatosis in that it decreased the level of adipose differentiation-related protein (P < 0.01) but increased mitochondrial proteins such as cytochrome c oxidase 4 and TFAM in GA muscle (P < 0.01). CONCLUSIONS: RORα plays a versatile role in regulating the quantity of mitochondria and the oxidative capacity, ultimately leading to an improvement in myosteatosis symptoms.

An RORα agonist, ODH-08, inhibits fibrogenic activation of hepatic stellate cells via suppression of SMAD3.

AIMS: Hepatic fibrosis is a dynamic process characterized by the net accumulation of an extracellular matrix resulting from chronic liver injury such as nonalcoholic steatohepatitis. Activation of hepatic stellate cells (HSCs) plays a role in transdifferentiation of quiescent cells into fibrogenic myofibroblasts. We aimed to examine the function of retinoic acid receptor-related orphan receptor alpha (RORα) and its novel agonistic ligand, 1-(4-benzyloxybenzyl)-3-(2-dimethylaminoethyl)-thiourea (ODH-08) against activation of HSCs using hepatic fibrosis mouse models. MAIN METHODS: Chemical synthesis, a reporter gene assay, surface plasmon resonance analysis, and a docking study were performed to evaluate ODH-08 as a ligand of RORα. In vivo experiments with mice fed a Western diet were performed to evaluate the effect of ODH-08. The human HSC line, Lx-2, and primary mouse HSCs were employed to identify the molecular mechanisms underlying the antifibrogenic effect of ODH-08. KEY FINDINGS: A novel RORα-selective ligand, ODH-08, was developed based on modification of JC1-40, an analog of N-methylthiourea. Administration of ODH-08 to the Western diet-fed mice reduced hepatic collagen deposition and expression levels of fibrogenic markers such as α-smooth muscle actin and collagen type I alpha 1 chain. Activation of RORα-either by transient overexpression of RORα or treatment with ODH-08-suppressed the expression of fibrogenic proteins in HSCs. The activation of RORα suppressed the activity of SMAD2 and 3, which are the primary downstream proteins of transforming growth factor ß. SIGNIFICANCE: RORα and its agonist ODH-08 have a potent antifibrotic effect, which could provide a novel antifibrotic strategy against hepatic fibrosis.

NR1D1 Stimulates Antitumor Immune Responses in Breast Cancer by Activating cGAS-STING Signaling.

Potentiating antitumor immunity is a promising therapeutic approach for treating a variety of cancers, including breast cancer. One potential strategy to promote antitumor immunity is targeting DNA damage response. Given that the nuclear receptor NR1D1 (also known as REV-ERBα) inhibits DNA repair in breast cancer cells, we explored the role of NR1D1 in antitumor CD8+ T-cell responses. First, deletion of Nr1d1 in MMTV-PyMT transgenic mice resulted in increased tumor growth and lung metastasis. Orthotopic allograft experiments suggested that loss of Nr1d1 in tumor cells rather than in stromal cells played a prominent role in increasing tumor progression. Comprehensive transcriptome analyses revealed that biological processes including type I IFN signaling and T cell-mediated immune responses were associated with NR1D1. Indeed, the expression of type I IFNs and infiltration of CD8+ T cells and natural killer cells in tumors were suppressed in Nr1d1-/-;MMTV-PyMT mice. Mechanistically, NR1D1 promoted DNA damage-induced accumulation of cytosolic DNA fragments and activated cGAS-STING signaling, which increased the production of type I IFNs and downstream chemokines CCL5 and CXCL10. Pharmacologic activation of NR1D1 by its ligand, SR9009, enhanced type I IFN-mediated antitumor immunity accompanied by the suppression of tumor progression and lung metastasis. Taken together, these findings reveal the critical role of NR1D1 in enhancing antitumor CD8+ T-cell responses, suggesting that NR1D1 may be a good therapeutic target for breast cancer. SIGNIFICANCE: NR1D1 suppresses breast cancer progression and lung metastasis by enhancing antitumor immunity via cGAS-STING pathway activation, which provides potential immunotherapeutic strategies for breast cancer.

Targeting HMG-CoA synthase 2 suppresses tamoxifen-resistant breast cancer growth by augmenting mitochondrial oxidative stress-mediated cell death.

AIMS: In this study, we aimed to investigate previously unrecognized lipid metabolic perturbations in tamoxifen-resistant breast cancer (BC) by conducting comprehensive metabolomics and transcriptomics analysis. We identified the role of 3-hydroxy-3-methylglutary-coenzyme-A-synthase 2 (HMGCS2), a key enzyme responsible for ketogenesis, in tamoxifen-resistant BC growth. MAIN METHODS: Comprehensive metabolomics (CE-TOFMS, LC-TOFMS) and transcriptiomics analysis were performed to characterize metabolic pathways in tamoxifen-resistant BC cells. The upregulation of HMGCS2 were verified thorugh immunohistochemistry (IHC) in clinical samples obtained from patients with recurrent BC. HMGCS2 inhibitor was discovered through surface plasmon resonance analysis, enzyme assay, and additional molecular docking studies. The effect of HMGCS2 suppression on tumor growth was studied thorugh BC xenograft model, and intratumoral lipid metabolites were analyzed via MALDI-TOFMS imaging. KEY FINDINGS: We revealed that the level of HMGCS2 was highly elevated in both tamoxifen-resistant T47D sublines (T47D/TR) and clinical refractory tumor specimens from patients with ER+ breast cancer, who had been treated with adjuvant tamoxifen. Suppression of HMGCS2 in T47D/TR resulted in the accumulation of mitochondrial reactive oxygen species (mtROS) and apoptotic cell death. Further, we identified alphitolic acid, a triterpenoid natural product, as a novel HMGCS2-specific inhibitor that elevated mtROS levels and drastically retarded the growth of T47D/TR in in vitro and in vivo experiments. SIGNIFICANCE: Enhanced ketogenesis with upregulation of HMGCS2 is a potential metabolic vulnerability of tamoxifen-resistant BC that offers a new therapeutic opportunity for treating patients with ER+ BC that are refractory to tamoxifen treatment.

Cholesterol sulfate inhibits osteoclast differentiation and survival by regulating the AMPK-Sirt1-NF-κB pathway.

Cholesterol sulfate (CS) is an activator of retinoic acid-related orphan receptor α (RORα). CS treatment or RORα overexpression attenuates osteoclastogenesis in a collagen-induced arthritis mouse model. However, the mechanism by which CS and RORα regulate osteoclast differentiation remains largely unknown. Thus, we aimed to investigate the role of CS and RORα in osteoclastogenesis and their underlying mechanism. CS inhibited osteoclast differentiation, but RORα deficiency did not affect osteoclast differentiation and CS-mediated inhibition of osteoclastogenesis. CS enhanced adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and sirtuin1 (Sirt1) activity, leading to nuclear factor-κB (NF-κB) inhibition by decreasing acetylation at Lys310 of p65. The NF-κB inhibition was restored by AMPK inhibitor, but the effects of CS on AMPK and NF-κB were not altered by RORα deficiency. CS also induced osteoclast apoptosis, which may be due to sustained AMPK activation and consequent NF-κB inhibition, and the effects of CS were significantly reversed by interleukin-1ß treatment. Collectively, these results indicate that CS inhibits osteoclast differentiation and survival by suppressing NF-κB via the AMPK-Sirt1 axis in a RORα-independent manner. Furthermore, CS protects against bone destruction in lipopolysaccharide- and ovariectomy-mediated bone loss mouse models, suggesting that CS is a useful therapeutic candidate for treating inflammation-induced bone diseases and postmenopausal osteoporosis.

IFI16/Ifi202 released from breast cancer induces secretion of inflammatory cytokines from macrophages and promotes tumor growth.

In tumor microenvironment (TME), macrophages trigger and maintain inflammatory responses that promoting tumor progression. Many cellular proteins are secreted from tumors and modulate their own TME by modulating macrophage phenotypes. Recently, we reported that interferon-γ-inducible protein 16 (IFI16), which was identified as an innate immune DNA sensor recognizing foreign DNA, triggered type Ⅰ interferon responses in breast cancer (BC). However, whether IFI16 was released from BC and affects TME has not been studied. Here, we report that IFI16 and its mouse homolog Ifi202 were released from BC cells, but not from normal epithelial cells. Ifi202 induced secretion of proinflammatory cytokines such as Interleukin (IL)-1ß, IL-6, and Tumor necrosis factor-α from macrophages via binding toll-like receptor 2 and activating downstream signaling pathway. Growth of allografted mouse BC 4T1 lacking Ifi202 was suppressed and accompanied with increased infiltration and cytotoxic activity of CD8+ T lymphocytes. Further, IFI16 was detected in sera of patients with BC. High expression level of IFI16 was associated with poor prognosis in patients with BC. Taken together, our findings suggest a novel role of IFI16/Ifi202 in TME, that elicits tumor promoting inflammation and thereby shaping immunosuppressive TME in BC.

Chemotactic cytokines secreted from Kupffer cells contribute to the sex-dependent susceptibility to non-alcoholic fatty liver diseases in mice.

AIMS: The global prevalence of non-alcoholic fatty liver disease (NAFLD) has rapidly increased over the last decade due to an elevated occurrence of metabolic syndromes. Importantly, the prevalence and severity of NAFLD is higher in men than in women. Therefore, in the present study we endeavored to identify the mechanistic disparity between male and female mice. MAIN METHODS: Global gene transcriptomics analysis was done with the high-fat diet (HFD)-induced NAFLD model of male, female, and ovariectomized (OVX) female mice. The expression of CCL2, CXCL2, and CXCL10 in mRNA level and serum protein level was done by qPCR and ELISA each. Immunohistochemistry staining was used to observe hepatic immune cell infiltration. To analyzing portion of immune cells, flow cytometry was done with isolated liver cells from HFD-fed male and female mice. Primary mouse liver cells were isolated from male and female mice for in vitro studies. KEY FINDINGS: We identified sex differences in inflammatory chemokines, CCL2, CXCL2, and CXCL10, with the expression of these chemokines enhanced in male and OVX, but not in female, mice after HFD feeding. Resident Kupffer cells (KCs) were identified as the major source of production of CCL2, CXCL2, and CXCL10 in the mouse NAFLD model. Notably, KCs obtained from male mice expressed higher levels of chemokines than those from female mice, indicating that KCs may mediate the sex discrepancy in NAFLD progression. SIGNIFICANCE: Our findings offer new insights into the pathology of sex-specific differences in NAFLD, involving chemokines and KCs.

Polyploidization of Hepatocytes: Insights into the Pathogenesis of Liver Diseases.

Polyploidization is a process by which cells are induced to possess more than two sets of chromosomes. Although polyploidization is not frequent in mammals, it is closely associated with development and differentiation of specific tissues and organs. The liver is one of the mammalian organs that displays ploidy dynamics in physiological homeostasis during its development. The ratio of polyploid hepatocytes increases significantly in response to hepatic injury from aging, viral infection, iron overload, surgical resection, or metabolic overload, such as that from non-alcoholic fatty liver diseases (NAFLDs). One of the unique features of NAFLD is the marked heterogeneity of hepatocyte nuclear size, which is strongly associated with an adverse liver-related outcome, such as hepatocellular carcinoma, liver transplantation, and liver-related death. Thus, hepatic polyploidization has been suggested as a potential driver in the progression of NAFLDs that are involved in the control of the multiple pathogenicity of the diseases. However, the importance of polyploidy in diverse pathophysiological contexts remains elusive. Recently, several studies reported successful improvement of symptoms of NAFLDs by reducing pathological polyploidy or by controlling cell cycle progression in animal models, suggesting that better understanding the mechanisms of pathological hepatic polyploidy may provide insights into the treatment of hepatic disorders.

Type I IFN stimulates IFI16-mediated aromatase expression in adipocytes that promotes E2-dependent growth of ER-positive breast cancer.

Although type I interferons (IFNs) play multifaceted roles during tumorigenesis and cancer treatment, the interplay between type I IFNs and estrogen signaling in breast cancer (BC) microenvironment is not well understood. Here, we report a novel function of type I IFNs in inducing aromatase expression in adipose tissues surrounding BC, which potentiates the E2-dependent growth of estrogen receptor (ER)-positive BC. First, we found that expression levels of type I IFNs correlate negatively with clinical outcome but positively with tumor grade in patients with ER-positive BC. Levels of type I IFNs were elevated in cocultured media of immune cells and BC cells, which increased aromatase expression and E2 production in Simpson-Golabi-Behmel syndrome preadipocytes. The type I IFN-induced aromatase expression was dependent on IFN-γ-inducible protein 16 (IFI16), which is encoded by an interferon-stimulated gene. At the molecular level, type I IFNs led to recruitment of HIF1α-IFI16-PRMT2 complex to the hypoxia-response element located in the aromatase PI.3/PII promoter. Next, we generated an adipocyte-specific Ifi204, which is a mouse ortholog of human IFI16, knockout mouse (Ifi204-AKO). IFNß induced E2 production in the preadipocytes isolated from the control mice, but such E2 production was far lower in the Ifi204-AKO preadipocytes. Importantly, the growth of orthotopically inoculated E0771 ER-positive mammary tumors was reduced significantly in the Ifi204-AKO mice. Taken together, our findings provide novel insights into the crosstalk between type I IFNs and estrogen signaling in the progression of ER-positive BC.

RORα contributes to the maintenance of genome ploidy in the liver of mice with diet-induced nonalcoholic steatohepatitis.

Hepatic polyploidization is closely linked to the progression of nonalcoholic fatty liver disease (NAFLD); however, the underlying molecular mechanism is not clearly understood. In this study, we demonstrated the role of retinoic acid-related orphan receptor α (RORα) in the maintenance of genomic integrity, particularly in the pathogenesis of NAFLD, using the high-fat diet (HFD)-fed liver-specific RORα knockout (RORα-LKO) mouse model. First, we observed that the loss of hepatic retinoic acid receptor-related orphan receptor α (RORα) accelerated hepatocyte nuclear polyploidization after HFD feeding. In 70% partial hepatectomy experiments, enrichment of hepatocyte polyploidy was more obvious in the RORα-LKO animals, which was accompanied by early progression to the S phase and blockade of the G2/M transition, suggesting a potential role of RORα in suppressing hepatocyte polyploidization in the regenerating liver. An analysis of a publicly available RNA sequencing (RNA-seq) and chromatin immunoprecipitation-seq dataset, together with the Search Tool of the Retrieval of Interacting Genes/Proteins database resource, revealed that DNA endoreplication was the top-enriched biological process Gene Ontology term. Furthermore, we found that E2f7 and E2f8, which encode key transcription factors for DNA endoreplication, were the downstream targets of RORα-induced transcriptional repression. Finally, we showed that the administration of JC1-40, an RORα activator (5 mg/kg body wt), significantly reduced hepatic nuclear polyploidization in the HFD-fed mice. Together, our observations suggest that the RORα-induced suppression of hepatic polyploidization may provide new insights into the pathological polyploidy of NAFLD and may contribute to the development of therapeutic strategies for the treatment of NAFLD.NEW & NOTEWORTHY It has been reported that hepatic polyploidization is closely linked to the progression of NAFLD. Here, we showed that the genetic depletion of hepatic RORα in mice accelerated hepatocyte polyploidization after high-fat diet feeding. The mechanism could be the RORα-mediated repression of E2f7 and E2f8, key transcription factors for DNA endoreplication. Thus, preservation of genome integrity by RORα could provide a new insight for developing therapeutics against the disease.

IFI16 inhibits DNA repair that potentiates type-I interferon-induced antitumor effects in triple negative breast cancer.

Tumor DNA-damage response (DDR) has an important role in driving type-I interferon (IFN)-mediated host antitumor immunity, but it is not clear how tumor DNA damage is interconnected with the immune response. Here, we report the role of IFN-γ-inducible protein 16 (IFI16) in DNA repair, which amplifies the stimulator of IFN genes (STING)-type-I IFN signaling, particularly in triple-negative breast cancer (TNBC). IFI16 is rapidly induced and accumulated to the histone-evicted DNA at double-stranded breakage (DSB) sites, where it inhibits recruitment of DDR factors. Subsequently, IFI16 increases the release of DNA fragments to the cytoplasm and induces STING-mediated type-I IFN production. Synergistic cytotoxic and immunomodulatory effects of doxorubicin and type-I IFNs are decreased upon IFI16 depletion in vivo. Furthermore, IFI16 expression correlates with improved clinical outcome in patients with TNBC treated with chemotherapy. Together, our findings suggest that type-I IFNs and IFI16 could offer potential therapeutic strategies for TNBC.

Tentonin 3/TMEM150C regulates glucose-stimulated insulin secretion in pancreatic ß-cells.

Glucose homeostasis is initially regulated by the pancreatic hormone insulin. Glucose-stimulated insulin secretion in ß-cells is composed of two cellular mechanisms: a high glucose concentration not only depolarizes the membrane potential of the ß-cells by ATP-sensitive K+ channels but also induces cell inflation, which is sufficient to release insulin granules. However, the molecular identity of the stretch-activated cation channel responsible for the latter pathway remains unknown. Here, we demonstrate that Tentonin 3/TMEM150C (TTN3), a mechanosensitive channel, contributes to glucose-stimulated insulin secretion by mediating cation influx. TTN3 is expressed specifically in ß-cells and mediates cation currents to glucose and hypotonic stimulations. The glucose-induced depolarization, firing activity, and Ca2+ influx of ß-cells were significantly lower in Ttn3-/- mice. More importantly, Ttn3-/- mice show impaired glucose tolerance with decreased insulin secretion in vivo. We propose that TTN3, as a stretch-activated cation channel, contributes to glucose-stimulated insulin secretion.

RORα Enhances Lysosomal Acidification and Autophagic Flux in the Hepatocytes.

Lysosomes are intracellular acidic organelles with catabolic functions that contribute to the activation of autophagy. Although autophagy abnormality is associated with defects in lysosomal acidification during the progression of nonalcoholic fatty liver disease (NAFLD), the mechanisms of control of lysosomal acidification are not well understood at the molecular level. Thus, we aimed to elucidate the role of the orphan nuclear receptor retinoic acid-related orphan receptor α (RORα) in lysosomal acidification and autophagic flux, particularly in nutrition-enriched hepatocytes. First, lysosomal acidity was much lower in the hepatocytes obtained from hepatocyte-specific RORα-deleted (RORα-LKO) mice, whereas the infusion of an adenovirus encoding RORα in wild-type hepatocytes increased lysosomal acidity, as determined by LysoSensor. Second, the lysosomal translocation of the mechanistic target of rapamycin was increased and immature cathepsin D was accumulated in the liver of RORα-LKO mice. Third, the accumulation of LC3-II, p62/sequestosome 1 (SQSTM1), and neighbor of BRCA1 gene 1 (NBR1) was increased in the livers of RORα-LKO mice, indicating an impaired autophagic flux in the livers. Consistently, the number of autolysosomes containing mitochondria and lipid droplets was dramatically reduced in the RORα-deleted hepatocytes. Finally, we found that RORα induced the transcription of genes involved in lysosomal function, such as Atp6v1g1, a vacuolar H+ -ATPase (v-ATPase) subunit, which were largely down-regulated in the livers of mice with high-fat diet-induced NAFLD and patients with hepatitis. Conclusion: Targeting RORα may be a potential therapeutic strategy to restore lysosomal acidification, which inhibits the progression of NAFLD.

Bioactive Lipids and Their Derivatives in Biomedical Applications.

Lipids, which along with carbohydrates and proteins are among the most important nutrients for the living organism, have a variety of biological functions that can be applied widely in biomedicine. A fatty acid, the most fundamental biological lipid, may be classified by length of its aliphatic chain, and the short-, medium-, and long-chain fatty acids and each have distinct biological activities with therapeutic relevance. For example, short-chain fatty acids have immune regulatory activities and could be useful against autoimmune disease; medium-chain fatty acids generate ketogenic metabolites and may be used to control seizure; and some metabolites oxidized from long-chain fatty acids could be used to treat metabolic disorders. Glycerolipids play important roles in pathological environments, such as those of cancers or metabolic disorders, and thus are regarded as a potential therapeutic target. Phospholipids represent the main building unit of the plasma membrane of cells, and play key roles in cellular signaling. Due to their physical properties, glycerophospholipids are frequently used as pharmaceutical ingredients, in addition to being potential novel drug targets for treating disease. Sphingolipids, which comprise another component of the plasma membrane, have their own distinct biological functions and have been investigated in nanotechnological applications such as drug delivery systems. Saccharolipids, which are derived from bacteria, have endotoxin effects that stimulate the immune system. Chemically modified saccharolipids might be useful for cancer immunotherapy or as vaccine adjuvants. This review will address the important biological function of several key lipids and offer critical insights into their potential therapeutic applications.

Specialized Proresolving Mediators for Therapeutic Interventions Targeting Metabolic and Inflammatory Disorders.

Uncontrolled inflammation is considered the pathophysiological basis of many prevalent metabolic disorders, such as nonalcoholic fatty liver disease, diabetes, obesity, and neurodegenerative diseases. The inflammatory response is a self-limiting process that produces a superfamily of chemical mediators, called specialized proresolving mediators (SPMs). SPMs include the ω-3-derived family of molecules, such as resolvins, protectins, and maresins, as well as arachidonic acid-derived (ω-6) lipoxins that stimulate and promote resolution of inflammation, clearance of microbes, and alleviation of pain and promote tissue regeneration via novel mechanisms. SPMs function by binding and activating G protein-coupled receptors, such as FPR2/ALX, GPR32, and ERV1, and nuclear orphan receptors, such as RORα. Recently, several studies reported that SPMs have the potential to attenuate lipid metabolism disorders. However, the understanding of pharmacological aspects of SPMs, including tissue-specific biosynthesis, and specific SPM receptors and signaling pathways, is currently limited. Here, we summarize recent advances in the role of SPMs in resolution of inflammatory diseases with metabolic disorders, such as nonalcoholic fatty liver disease and obesity, obtained from preclinical animal studies. In addition, the known SPM receptors and their intracellular signaling are reviewed as targets of resolution of inflammation, and the currently available information on the therapeutic effects of major SPMs for metabolic disorders is summarized.

STK3/STK4 signalling in adipocytes regulates mitophagy and energy expenditure.

Obesity reduces adipocyte mitochondrial function, and expanding adipocyte oxidative capacity is an emerging strategy to improve systemic metabolism. Here, we report that serine/threonine-protein kinase 3 (STK3) and STK4 are key physiological suppressors of mitochondrial capacity in brown, beige and white adipose tissues. Levels of STK3 and STK4, kinases in the Hippo signalling pathway, are greater in white than brown adipose tissues, and levels in brown adipose tissue are suppressed by cold exposure and greatly elevated by surgical denervation. Genetic inactivation of Stk3 and Stk4 increases mitochondrial mass and function, stabilizes uncoupling protein 1 in beige adipose tissue and confers resistance to metabolic dysfunction induced by high-fat diet feeding. Mechanistically, STK3 and STK4 increase adipocyte mitophagy in part by regulating the phosphorylation and dimerization status of the mitophagy receptor BNIP3. STK3 and STK4 expression levels are elevated in human obesity, and pharmacological inhibition improves metabolic profiles in a mouse model of obesity, suggesting STK3 and STK4 as potential targets for treating obesity-related diseases.

Ahnak deficiency attenuates high-fat diet-induced fatty liver in mice through FGF21 induction.

The AHNAK nucleoprotein has been determined to exert an anti-obesity effect in adipose tissue and further inhibit adipogenic differentiation. In this study, we examined the role of AHNAK in regulating hepatic lipid metabolism to prevent diet-induced fatty liver. Ahnak KO mice have reportedly exhibited reduced fat accumulation in the liver and decreased serum triglyceride (TG) levels when provided with either a normal chow diet or a high-fat diet (HFD). Gene expression profiling was used to identify novel factors that could be modulated by genetic manipulation of the Ahnak gene. The results revealed that fibroblast growth factor 21 (FGF21) was markedly increased in the livers of Ahnak KO mice compared with WT mice fed a HFD. Ahnak knockdown in hepatocytes reportedly prevented excessive lipid accumulation induced by palmitate treatment and was associated with increased secretion of FGF21 and the expression of genes involved in fatty acid oxidation, which are primarily downstream of PPARα. These results indicate that pronounced obesity and hepatic steatosis are attenuated in HFD-fed Ahnak KO mice. This may be attributed, in part, to the induction of FGF21 and regulation of lipid metabolism, which are considered to be involved in increased fatty acid oxidation and reduced lipogenesis in the liver. These findings suggest that targeting AHNAK may have beneficial implications in preventing or treating hepatic steatosis.

A critical regulation of Th2 cell responses by RORα in allergic asthma.

Allergic asthma is a chronic inflammatory disease of the lung and the airway, which is characterized by aberrant type 2 immune responses to otherwise unharmful aeroallergens. While the central role of Th2 cells and type 2 cytokines in the pathogenesis of allergic asthma is well documented, the regulation and plasticity of Th2 cells remain incompletely understood. By using an animal model of allergic asthma in IL-4-reporter mice, we found that Th2 cells in the lung expressed higher levels of Rora than those in the lymph nodes, and that treatment with an RORα agonist SR1078 resulted in diminished Th2 cell responses in vivo. To determine the T cell-intrinsic role of RORα in allergic asthma in vivo, we established T cell-specific RORα-deficient (Cd4creRoraf/f) mice. Upon intranasal allergen challenges, Cd4creRoraf/f mice exhibited a significantly increased Th2 cells in the lungs and the airway and showed an enhanced eosinophilic inflammation compared to littermate control mice. Studies with Foxp3YFP-creRoraf/f mice and CD8+ T cell depletion showed that the increased Th2 cell responses in the Cd4creRoraf/f mice were independent of Treg cells and CD8+ T cells. Our findings demonstrate a critical regulatory role of RORα in Th2 cells, which suggest that RORα agonists could be effective for the treatment of allergic diseases.

RORα regulates hepatic lipolysis by inducing transcriptional expression of PNPLA3 in mice.

Nonalcoholic fatty liver diseases (NAFLDs) are characterized by excessive triacylglycerol (TAG) accumulation in the liver which contributes to hepatocyte dysfunction, inflammation, and fibrosis. Patatin-like phospholipase domain-containing 3 (PNPLA3; also known as adiponutrin) has emerged as an important enzyme leading to hepatic TAG hydrolysis. Because the I148M substitution in the PNPLA3 gene markedly reduces hepatic TAG hydrolase activity, this genetic variation is strongly associated with increased hepatic TAG in the full spectrum of NAFLDs. The Retinoic acid-related orphan receptor α (RORα) regulates various target genes related to lipid metabolism. Here, we investigated the role of RORα on PNPLA3-mediated hepatic lipid hydrolysis. With blockade of lipid esterification and ß-oxidation, RORα enhanced TAG hydrolysis, resulting in increased free glycerol levels. We found a putative RORα response element on the upstream of PNPLA3 gene that was activated by RORα. Furthermore, the inhibitory action of cJUN on the RORα/PNPLA3 axis was enhanced under lipid stress and contributed to hepatic lipid accumulation. In summary, we showed for the first time that RORα activates the transcription of PNPLA3, which suggests that RORα and its ligands represent potential precision therapeutic approaches for NAFLDs.