Novel insights into regulators and functional modulators of adipogenesis.

Adipogenesis is a process that differentiates new adipocytes from precursor cells and is tightly regulated by several factors, including many transcription factors and various post-translational modifications. Recently, new roles of adipogenesis have been suggested in various diseases. However, the molecular mechanisms and functional modulation of these adipogenic genes remain poorly understood. This review summarizes the regulatory factors and modulators of adipogenesis and discusses future research directions to identify novel mechanisms regulating adipogenesis and the effects of adipogenic regulators in pathological conditions. The master adipogenic transcriptional factors PPARγ and C/EBPα were identified along with other crucial regulatory factors such as SREBP, Kroxs, STAT5, Wnt, FOXO1, SWI/SNF, KLFs, and PARPs. These transcriptional factors regulate adipogenesis through specific mechanisms, depending on the adipogenic stage. However, further studies related to the in vivo role of newly discovered adipogenic regulators and their function in various diseases are needed to develop new potent therapeutic strategies for metabolic diseases and cancer.

Release of ATP in the Lung Evoked by Inhalation of Irritant Gases in Rats.

Adenosine triphosphate (ATP) can be released into the extracellular milieu from various types of cells in response to a wide range of physical or chemical stresses. In the respiratory tract, extracellular ATP is recognized as an important signal molecule and trigger of airway inflammation. Chlorine (Cl2), sulfur dioxide (SO2), and ammonia (NH3) are potent irritant gases and common industrial air pollutants due to their widespread uses as chemical agents. This study was carried out to determine if acute inhalation challenges of these irritant gases, at the concentration and duration simulating the accidental exposures to these chemical gases in industrial operations, triggered the release of ATP in the rat respiratory tract; and if so, whether the level of ATP in bronchoalveolar lavage fluid (BALF) evoked by inhalation challenge of a given irritant gas was elevated by chronic allergic airway inflammation. Our results showed: 1) Inhalation of these irritant gases caused significant increases in the ATP level in BALF, and the magnitude of evoked ATP release was in the order of Cl2 > SO2 > NH3. 2) Chronic airway inflammation induced by ovalbumin-sensitization markedly elevated the ATP level in BALF during baseline (breathing room air) but did not potentiate the release of ATP in the lung triggered by inhalation challenges of these irritant gases. These findings suggested a possible involvement of the ATP release in the lung in the regulation of overall airway responses to acute inhalation of irritant gases and the pathogenesis of chronic allergic airway inflammation.

Macrophage-restricted overexpression of glutaredoxin 1 protects against atherosclerosis by preventing nutrient stress-induced macrophage dysfunction and reprogramming.

BACKGROUND AND AIMS: Deficiency in the thiol transferase glutaredoxin 1 (Grx1) in aging mice promotes, in a sexually dimorphic manner, dysregulation of macrophages and atherogenesis. However, the underlying mechanisms are not known. Here we tested the hypothesis that macrophage-restricted overexpression of Grx1 protects atherosclerosis-prone mice against macrophage reprogramming and dysfunction induced by a high-calorie diet (HCD) and thereby reduces the severity of atherosclerosis. METHODS: We generated lentiviral vectors carrying cluster of differentiation 68 (CD68) promoter-driven enhanced green fluorescent protein (EGFP) or Grx1 constructs and conducted bone marrow (BM) transplantation studies to overexpress Grx1 in a macrophage-specific manner in male and female atherosclerosis-prone LDLR-/- mice, and fed these mice a HCD to induce atherogenesis. Atherosclerotic lesion size was determined in both the aortic root and the aorta. We isolated BM-derived macrophages (BMDM) to assess protein S-glutathionylation levels and loss of mitogen-activated protein kinase phosphatase 1 (MKP-1) activity as measures of HCD-induced thiol oxidative stress. We also conducted gene profiling on these BMDM to determine the impact of Grx1 activity on HCD-induced macrophage reprogramming. RESULTS: Overexpression of Grx1 protected macrophages against HCD-induced protein S-glutathionylation, reduced monocyte chemotaxis in vivo, limited macrophage recruitment into atherosclerotic lesions, and was sufficient to reduce the severity of atherogenesis in both male and female mice. Gene profiling revealed major sex differences in the transcriptional reprogramming of macrophages induced by HCD feeding, but Grx1 overexpression only partially reversed HCD-induced transcriptional reprogramming of macrophages. CONCLUSIONS: Macrophage Grx1 plays a major role in protecting mice atherosclerosis mainly by maintaining the thiol redox state of the macrophage proteome and preventing macrophage dysfunction.

Inhibiting 5-lipoxygenase prevents skeletal muscle atrophy by targeting organogenesis signalling and insulin-like growth factor-1.

BACKGROUND: Skeletal muscle atrophy can occur in response to numerous factors, such as ageing and certain medications, and produces a major socio-economic burden. At present, there are no approved drugs for treating skeletal muscle atrophy. Arachidonate 5-lipoxygenase (Alox5) is a drug target for a number of diseases. However, pharmacological targeting of Alox5, and its role in skeletal muscle atrophy, is unclear. METHODS: The potential effects of gene knockdown and pharmacological targeting of Alox5 on skeletal muscle atrophy were investigated using cell-based models, animal models and human skeletal muscle primary cells. Malotilate, a clinically safe drug developed for enhancing liver regeneration and Alox5 inhibitor, was investigated as a repurposing candidate. Mechanism(s) of action in skeletal muscle atrophy was assessed by measuring the expression level or activation status of key regulatory pathways and validated using gene knockdown and RNA sequencing. RESULTS: Myotubes treated with the atrophy-inducing glucocorticoid, dexamethasone, were protected from catabolic responses by treatment with malotilate (+41.29%, P < 0.01). Similar anti-atrophy effects were achieved by gene knockdown of Alox5 (+30.4%, P < 0.05). Malotilate produced anti-atrophy effects without affecting the myogenic differentiation programme. In an in vivo model of skeletal muscle atrophy, malotilate treatment preserved muscle force/strength (grip strength: +35.72%, latency to fall: +553.1%, P < 0.05), increased mass and fibre cross-sectional area (quadriceps: +23.72%, soleus: +33.3%, P < 0.01) and down-regulated atrogene expression (Atrogin-1: -61.58%, Murf-1: -66.06%, P < 0.01). Similar, beneficial effects of malotilate treatment were observed in an ageing muscle model, which also showed the preservation of fast-twitch fibres (Type 2a: +56.48%, Type 2b: +37.32%, P < 0.01). Leukotriene B4, a product of Alox5 activity with inflammatory and catabolic functions, was found to be elevated in skeletal muscle undergoing atrophy (quadriceps: +224.4%, P < 0.001). Cellular transcriptome analysis showed that targeting Alox5 up-regulated biological processes regulating organogenesis and increased the expression of insulin-like growth factor-1, a key anti-atrophy hormone (+226.5%, P < 0.05). Interestingly, these effects were restricted to the atrophy condition and not observed in normal skeletal muscle cultures with Alox5 inhibition. Human myotubes were also protected from atrophy by pharmacological targeting of Alox5 (+23.68%, P < 0.05). CONCLUSIONS: These results shed new light on novel drug targets and mechanisms underpinning skeletal muscle atrophy. Alox5 is a regulator and drug target for muscle atrophy, and malotilate is an attractive compound for repurposing studies to treat this disease.

Investigation of niclosamide as a repurposing agent for skeletal muscle atrophy.

Skeletal muscle atrophy is a feature of aging (termed sarcopenia) and various diseases, such as cancer and kidney failure. Effective drug treatment options for muscle atrophy are lacking. The tapeworm medication, niclosamide is being assessed for repurposing to treat numerous diseases, including end-stage cancer metastasis and hepatic steatosis. In this study, we investigated the potential of niclosamide as a repurposing drug for muscle atrophy. In a myotube atrophy model using the glucocorticoid, dexamethasone, niclosamide did not prevent the reduction in myotube diameter or the decreased expression of phosphorylated FOXO3a, which upregulates the ubiquitin-proteasome pathway of muscle catabolism. Treatment of normal myotubes with niclosamide did not activate mTOR, a major regulator of muscle protein synthesis, and increased the expression of atrogin-1, which is induced in catabolic states. Niclosamide treatment also inhibited myogenesis in muscle precursor cells, enhanced the expression of myoblast markers Pax7 and Myf5, and downregulated the expression of differentiation markers MyoD, MyoG and Myh2. In an animal model of muscle atrophy, niclosamide did not improve muscle mass, grip strength or muscle fiber cross-sectional area. Muscle atrophy is also feature of cancer cachexia. IC50 analyses indicated that niclosamide was more cytotoxic for myoblasts than cancer cells. In addition, niclosamide did not suppress the induction of iNOS, a key mediator of atrophy, in an in vitro model of cancer cachexia and did not rescue myotube diameter. Overall, these results suggest that niclosamide may not be a suitable repurposing drug for glucocorticoid-induced skeletal muscle atrophy or cancer cachexia. Nevertheless, niclosamide may be employed as a compound to study mechanisms regulating myogenesis and catabolic pathways in skeletal muscle.

Lithium Chloride Protects against Sepsis-Induced Skeletal Muscle Atrophy and Cancer Cachexia.

Inflammation-mediated skeletal muscle wasting occurs in patients with sepsis and cancer cachexia. Both conditions severely affect patient morbidity and mortality. Lithium chloride has previously been shown to enhance myogenesis and prevent certain forms of muscular dystrophy. However, to our knowledge, the effect of lithium chloride treatment on sepsis-induced muscle atrophy and cancer cachexia has not yet been investigated. In this study, we aimed to examine the effects of lithium chloride using in vitro and in vivo models of cancer cachexia and sepsis. Lithium chloride prevented wasting in myotubes cultured with cancer cell-conditioned media, maintained the expression of the muscle fiber contractile protein, myosin heavy chain 2, and inhibited the upregulation of the E3 ubiquitin ligase, Atrogin-1. In addition, it inhibited the upregulation of inflammation-associated cytokines in macrophages treated with lipopolysaccharide. In the animal model of sepsis, lithium chloride treatment improved body weight, increased muscle mass, preserved the survival of larger fibers, and decreased the expression of muscle-wasting effector genes. In a model of cancer cachexia, lithium chloride increased muscle mass, enhanced muscle strength, and increased fiber cross-sectional area, with no significant effect on tumor mass. These results indicate that lithium chloride exerts therapeutic effects on inflammation-mediated skeletal muscle wasting, such as sepsis-induced muscle atrophy and cancer cachexia.

Screening ginseng saponins in progenitor cells identifies 20(R)-ginsenoside Rh2 as an enhancer of skeletal and cardiac muscle regeneration.

Aging is associated with increased prevalence of skeletal and cardiac muscle disorders, such as sarcopenia and cardiac infarction. In this study, we constructed a compendium of purified ginsenoside compounds from Panax ginseng C.A. Meyer, which is a traditional Korean medicinal plant used to treat for muscle weakness. Skeletal muscle progenitor cell-based screening identified three compounds that enhance cell viability, of which 20(R)-ginsenoside Rh2 showed the most robust response. 20(R)-ginsenoside Rh2 increased viability in myoblasts and cardiomyocytes, but not fibroblasts or disease-related cells. The cellular mechanism was identified as downregulation of cyclin-dependent kinase inhibitor 1B (p27Kip1) via upregulation of Akt1/PKB phosphorylation at serine 473, with the orientation of the 20 carbon epimer being crucially important for biological activity. In zebrafish and mammalian models, 20(R)-ginsenoside Rh2 enhanced muscle cell proliferation and accelerated recovery from degeneration. Thus, we have identified 20(R)-ginsenoside Rh2 as a p27Kip1 inhibitor that may be developed as a natural therapeutic for muscle degeneration.

Role of Heparin-Binding Epidermal Growth Factor-Like Growth Factor in Oxidative Stress-Associated Metabolic Diseases.

Heparin-binding EGF-like growth factor (HB-EGF) is an EGF family member that interacts with epidermal growth factor receptor (EGFR) and ERBB4. Since HB-EGF was first identified as a novel growth factor secreted from a human macrophage cell line, numerous pathological and physiological functions related to cell proliferation, migration, and inflammation have been reported. Notably, the expression of HB-EGF is sensitively upregulated by oxidative stress in the endothelial cells and functions for auto- and paracrine-EGFR signaling. Overnutrition and obesity cause elevation of HB-EGF expression and EGFR signaling in the hepatic and vascular systems. Modulations of HB-EGF signaling showed a series of protections against phenotypes related to metabolic syndrome and advanced metabolic diseases, suggesting HB-EGF as a potential target against metabolic diseases.

Anti-oxidant activity reinforced reduced graphene oxide/alginate microgels: Mesenchymal stem cell encapsulation and regeneration of infarcted hearts.

Mesenchymal stem cell (MSC) transplantation is promising for repairing heart tissues post myocardial infarction (MI). In particular, paracrine effects of the transplanted MSCs have been highlighted to play major roles in heart regeneration by secreting multiple growth factors and immune-modulatory cytokines. Nevertheless, its therapeutic efficacy still remains low, which is strongly associated with low viability and activity of the transplanted stem cells, because the transplanted MSCs are exposed to high shear stress during injection and harsh environments (e.g., high oxidative stress and host immune reactions) post injection. In this study, we aimed to develop novel injectable MSC-delivery microgel systems possessing high anti-oxidant activities. Specifically, we encapsulated MSCs in graphene oxide (GO)/alginate composite microgels by electrospraying. To further enhance the anti-oxidizing activities of the gels, we developed reduced MSC-embedded GO/alginate microgels (i.e., r(GO/alginate)), which have the potential to protect MSCs from the abovementioned harsh environments within MI tissues. Our in vitro studies demonstrated that the MSCs encapsulated in the r(GO/alginate) microgels showed increased viability under oxidative stress conditions with H2O2. Furthermore, cardiomyocytes (CMs), co-cultured with the encapsulated MSCs in transwells with H2O2 treatment, showed higher cell viability and cardiac maturation compared to monolayer cultured CMs, likely due to ROS scavenging by the gels and positive paracrine signals from the encapsulated MSCs. In vivo experiments with acute MI models demonstrated improved therapeutic efficacy of MSC delivery in r(GO/alginate) microgels, exhibiting significant decreases in the infarction area and the improvement of cardiac function. We believe that our novel MSC encapsulation system with GO, alginate, and mild reduction, which exhibits high cell protection capacity (e.g., anti-oxidant activity), will serve as an effective platform for the delivery of stem cells and other therapeutic cell types to treat various injuries and diseases, including MI.

Chemical characterization and biological activity data for a novel indirubin derivative, LDD-1819.

This article contains chemical characterization and biological activity data for a novel indirubin derivative, termed LDD-1819. The detailed synthesis procedure and associated NMR data are presented. The concentration-dependent inhibition data of two biological targets, glycogen synthase kinase-3 ß and aurora kinase A are described. The following biological data are also contained in this article: 1) the cellularization of skeletal muscle myotubes by LDD-1819 or two small molecule inhibitors of glycogen synthase kinase-3 ß and aurora kinase A (BIO and reversine) and gene expression data for the myoblast markers Pax-7 and Myf5, 2) Cell viability of hTERT human immortalized fibroblasts, colon cancer cells and breast cancer cells, and 3) Western blotting analysis of full length and cleaved caspse-7, and cleaved poly (ADP-ribose) polymerase (PARP) in hTERT fibroblasts treated with LDD-1819. A schematic diagram of the biological activities of LDD-1819 is also presented. Further interpretation and discussion of these data are provided in the associated research article 'A novel indirubin derivative that increases somatic cell plasticity and inhibits tumorigenicity' (Kim et al., 2019).

A novel indirubin derivative that increases somatic cell plasticity and inhibits tumorigenicity.

Indirubin-based compounds affect diverse biological processes, such as inflammation and angiogenesis. In this study, we tested a novel indirubin derivative, LDD-1819 (2-((((2Z,3E)-5-hydroxy-5'-nitro-2'-oxo-[2,3'-biindolinylidene]-3-ylidene)amino)oxy)ethan-1-aminium chloride) for two major biological activities: cell plasticity and anti-cancer activity. Biological assays indicated that LDD-1819 induced somatic cell plasticity. LDD-1819 potentiated myoblast reprogramming into osteogenic cells and fibroblast reprogramming into adipogenic cells. Interestingly, in an assay of skeletal muscle dedifferentiation, LDD-1819 induced human muscle cellularization and blocked residual proliferative activity to produce a population of mononuclear refractory cells, which is also observed in the early stages of limb regeneration in urodele amphibians. In cancer cell lines, LDD-1819 treatment inhibited cell invasion and selectively induced apoptosis compared to normal cells. In an animal tumor xenograft model, LDD-1819 reduced human cancer cell metastasis in vivo at doses that did not produce toxicity. Biochemical assays showed that LDD-1819 possessed inhibitory activity against glycogen synthase kinase-3ß, which is linked to cell plasticity, and aurora kinase, which regulates carcinogenesis. These results indicate that novel indirubin derivative LDD-1819 is a dual inhibitor of glycogen synthase kinase-3ß and aurora A kinase, and has potential for development as an anti-cancer drug or as a reprogramming agent for cell-therapy based approaches to treat degenerative diseases.

Cancer-Stimulated CAFs Enhance Monocyte Differentiation and Protumoral TAM Activation via IL6 and GM-CSF Secretion.

Purpose: M2-type TAMs are increasingly implicated as a crucial factor promoting metastasis. Numerous cell types dictate monocyte differentiation into M2 TAMs via a complex network of cytokine-based communication. Elucidating critical pathways in this network can provide new targets for inhibiting metastasis. In this study, we focused on cancer cells, CAFs, and monocytes as a major node in this network.Experimental Design: Monocyte cocultures with cancer-stimulated CAFs were used to investigate differentiation into M2-like TAMs. Cytokine array analyses were employed to discover the CAF-derived regulators of differentiation. These regulators were validated in primary CAFs and bone marrow-derived monocytes. Orthotopic, syngeneic colon carcinoma models using cotransplanted CAFs were established to observe effects on tumor growth and metastasis. To confirm a correlation with clinical evidence, meta-analyses were employed using the Oncomine database.Results: Our coculture studies identify IL6 and GM-CSF as the pivotal signals released from cancer cell-activated CAFs that cooperate to induce monocyte differentiation into M2-like TAMs. In orthotopic, syngeneic colon carcinoma mouse models, cotransplanted CAFs elevated IL6 and GM-CSF levels, TAM infiltration, and metastasis. These pathologic effects were dramatically reversed by joint IL6 and GM-CSF blockade. A positive correlation between GM-CSF and IL6 expression and disease course was observed by meta-analyses of the clinical data.Conclusions: Our studies indicate a significant reappraisal of the role of IL6 and GM-CSF in metastasis and implicate CAFs as the "henchmen" for cancer cells in producing an immunosuppressive tumor ecological niche. Dual targeting of GM-CSF and IL6 is a promising new approach for inhibiting metastasis. Clin Cancer Res; 24(21); 5407-21. ©2018 AACR.

Targeting hepatic heparin-binding EGF-like growth factor (HB-EGF) induces anti-hyperlipidemia leading to reduction of angiotensin II-induced aneurysm development.

OBJECTIVE: The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model. APPROACH AND RESULTS: Low-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate. CONCLUSION: This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting.

Inhibition of natural gas hydrates in the presence of liquid hydrocarbons forming structure H.

The effects of LMGS (large molecule guest substance) amount on the thermodynamics of natural gas hydrates, as well as structural characteristics of mixed hydrates of LMGS and natural gas, have been studied. The addition of 1.7 wt % neohexane (NH) to water induced inhibition of natural gas hydrates, and this inhibition effect increased with increased addition of NH up to 7.8 wt %. However, the hydrate equilibrium condition changed slightly when the concentration of NH further increased from 7.8 to 14.5 wt %. Investigations on structural characteristics were carried out by analyzing (13)C NMR spectra of mixed hydrates formed from the mixture of natural gas and NH. They indicate that two hydrate structures of II and H coexist simultaneously, and the ratio of structure H to II decreased from 0.97 to 0.43 when the NH concentration decreased from 14.5 to 7.8 wt %. In addition, it was confirmed that ethane, propane, and iso-butane gas molecules do not participate in the formation of structure H and only enclathrated in large cages of structure II. These results indicate the existence of multiple hydrate structures, which must be considered in many industrial applications when mixed hydrates are formed from multicomponent gas mixtures and liquid hydrocarbons.