Bioactive Compounds and Adipocyte Browning Phenomenon

Overweight and obesity have become worldwide health issues in most countries. Current strategies aimed to prevent or reduce overweight and obesity have mainly focused on the genes and molecular mechanisms that give the functional characteristics to different types of adipose tissue. The Browning phenomenon in adipocytes consists of phenotypic and metabolic changes within white adipose tissue (WAT) activated by thermogenic mechanisms similar to that occurring in brown adipose tissue (BAT); this phenomenon has assumed great relevance due to its therapeutic potential against overweight and obesity. In addition, the study of inflammation in the development of overweight and obesity has also been included as a relevant factor, such as the pro-inflammatory mechanisms promoted by M1-type macrophages in adipose tissue. Studies carried out in this area are mainly performed by using the 3T3-L1 pre-adipocyte cell line, testing different bioactive compound sources such as plants and foods; nevertheless, it is necessary to standardize protocols used in vitro as well to properly scale them to animal models and clinical tests in order to have a better understanding of the mechanisms involved in overweight and obesity.     

CDK1 Inactivation Regulates Anaphase Spindle Dynamics and Cytokinesis In Vivo

Through association with CDK1, cyclin B accumulation and destruction govern the G₂/M/G₁ transitions in eukaryotic cells. To identify CDK1 inactivation-dependent events during late mitosis, we expressed a nondestructible form of cyclin B (cyclin BΔ90) by microinjecting its mRNA into prometaphase normal rat kidney cells. The injection inhibited chromosome decondensation and nuclear envelope formation. Chromosome disjunction occurred normally, but anaphase-like movement persisted until the chromosomes reached the cell periphery, whereupon they often somersaulted and returned to the cell center. Injection of rhodamine-tubulin showed that this movement occurred in the absence of a central anaphase spindle. In 82% of cells cytokinesis was inhibited; the remainder split themselves into two parts in a process reminiscent of Dictyostelium cytofission. In all cells injected, F-actin and myosin II were diffusely localized with no detectable organization at the equator. Our results suggest that a primary effect of CDK1 inactivation is on spindle dynamics that regulate chromosome movement and cytokinesis. Prolonged CDK1 activity may prevent cytokinesis through inhibiting midzone microtubule formation, the behavior of proteins such as TD60, or through the phosphorylation of myosin II regulatory light chain.     

Role of miR-1 and miR-133a in myocardial ischemic postconditioning

Background  

Ischemic postconditioning (IPost) has aroused much attention since 2003 when it was firstly reported. The role of microRNAs (miRNAs or miRs) in IPost has rarely been reported. The present study was undertaken to investigate whether miRNAs were involved in the protective effect of IPost against myocardial ischemia-reperfusion (IR) injury and the probable mechanisms involved.     

Insulin Regulates Adipocyte Lipolysis via an Akt-Independent Signaling Pathway

After a meal, insulin suppresses lipolysis through the activation of its downstream kinase, Akt, resulting in the inhibition of protein kinase A (PKA), the main positive effector of lipolysis. During insulin resistance, this process is ineffective, leading to a characteristic dyslipidemia and the worsening of impaired insulin action and obesity. Here, we describe a noncanonical Akt-independent, phosphoinositide-3 kinase (PI3K)-dependent pathway that regulates adipocyte lipolysis using restricted subcellular signaling. This pathway selectively alters the PKA phosphorylation of its major lipid droplet-associated substrate, perilipin. In contrast, the phosphorylation of another PKA substrate, hormone-sensitive lipase (HSL), remains Akt dependent. Furthermore, insulin regulates total PKA activity in an Akt-dependent manner. These findings indicate that localized changes in insulin action are responsible for the differential phosphorylation of PKA substrates. Thus, we identify a pathway by which insulin regulates lipolysis through the spatially compartmentalized modulation of PKA.The storage and mobilization of nutrients from specialized tissues requires the spatial organization of both signaling functions and energy stores. Nowhere is this more evident than in mammalian adipose tissue, which maintains the most efficient repository for readily available energy. Here, fuel is segregated into lipid droplets, once thought to be inert storehouses but now recognized as complex structures that represent a regulatable adaptation of a ubiquitous organelle (5, 40). The synthesis and maintenance of functional lipid droplets requires numerous proteins, not only fatty acid binding proteins and enzymes of lipid synthesis but also molecules critical to constitutive and specialized membrane protein trafficking (23).During times of nutritional need, triglycerides within the adipocyte lipid droplet are hydrolyzed into their components, fatty acids, acyl-glycerides, and, ultimately, glycerol. This process, termed lipolysis, is controlled dynamically by multiple hormonal signals that respond to the nutrient status of the organism. During fasting, catecholamines such as norepinephrine stimulate lipolysis via beta-adrenergic receptor activation, promoting adenylyl cyclase activity and the production of cyclic AMP (cAMP) (17). cAMP binds to the regulatory subunits of its major effector, protein kinase A (PKA), triggering the dissociation of these subunits and the subsequent activation of the catalytic subunits (62, 63). PKA is frequently sequestered into multiple parallel, intracellular signaling complexes, though such structures have not been studied in hormone-responsive adipocytes (68). Two targets of activated PKA important for lipolysis are hormone-sensitive lipase (HSL) and perilipin, the major lipid droplet coat protein (17). The phosphorylation of HSL on Ser 559/660 is crucial for its activation and translocation to the lipid droplet, where HSL catalyzes the hydrolysis of diglycerides to monoglycerides (26, 55). Another lipase, adipose triglyceride lipase (ATGL), carries out the initial cleavage of triglycerides to diglycerides and most likely is rate limiting for lipolysis, but it does not appear to be regulated directly via PKA phosphorylation (24, 73). Perilipin under basal conditions acts as a protective barrier against lipase activity; upon stimulation, the phosphorylation of least six PKA consensus sites triggers a conformational change in perilipin, permitting access to the lipid substrates in the droplet, the recruitment of HSL, and possibly the activation of ATGL (7, 8, 21, 41, 46, 58, 60, 61). Perilipin, therefore, possesses dual functions, both blocking lipolysis in the basal state as well as promoting lipolysis upon its phosphorylation (5, 58, 60).Following the ingestion of a meal, insulin stimulates the uptake of nutrients such as glucose into specialized tissues and also potently inhibits lipolysis in adipocytes (17). Insulin signaling in the adipocyte involves the activation of the insulin receptor tyrosine kinase, the phosphorylation of insulin receptor substrates, the activation of PI3K, and the subsequent production of specific phosphoinositides at the plasma membrane (59). These phosphoinositides then recruit Akt, via its pleckstrin homology domain, to the plasma membrane, where Akt becomes phosphorylated and activated by two upstream kinases. Akt stimulates the translocation of the glucose transporter GLUT4 to the plasma membrane, thereby promoting the uptake of glucose into the cell (2). The mechanism by which insulin inhibits lipolysis has been proposed to involve the reduction of cAMP levels and thus PKA activity. In this model, insulin signaling activates phosphodiesterase 3b (PDE3b) via the Akt-mediated phosphorylation of Ser273 (14, 32). Upon activation by Akt, PDE3b catalyzes the hydrolysis of cAMP to 5′AMP, thereby attenuating PKA activity and lipolysis. Recent studies of PDE3b knockout mice have highlighted the importance of PDE3b activity in the regulation of lipolysis but were uninformative regarding the mechanism of insulin action (12). Adipocytes isolated from these mice exhibit reduced responses to insulin with respect to lipolysis, but it is not clear whether this is due to the loss of the critical target enzyme or a normal mechanism being overwhelmed by supraphysiological concentrations of cAMP (12). Biochemical studies using dominant-inhibitory Akt have demonstrated that Akt can regulate PDE3b activity, and other studies also have suggested that Akt interacts directly with PDE3b, implying a direct connection to lipolysis regulation (1, 32). Nevertheless, the actual requirement for Akt in insulin action with regard to the lipolysis itself has not been demonstrated directly in, for example, genetic loss-of-function experiments.There now is substantial evidence implicating elevated free fatty acid levels as a consequence of inappropriate lipolysis as a major etiological factor for insulin resistance and type 2 diabetes mellitus (T2DM) (51). Conditions such as obesity and diabetes are characterized by a pathophysiological state in which these tissues become unresponsive to insulin, which contribute to the adverse long-term sequelae of diseases such as T2DM and the metabolic syndrome (4, 44). Thus, understanding in detail the mechanism by which insulin suppresses fat cell lipolysis is critical to identifying the underlying defect in resistant adipose tissue and ultimately developing effective therapeutics. In the present study, we investigated both Akt-dependent and -independent modes of insulin action toward lipolysis. We found the latter to predominate at low, physiological levels of adrenergic stimulation, acting via a pathway dependent on the preferential phosphorylation of downstream PKA substrates.     

Circulating miR-1, miR-133a,and miR-206 levels are increased after a half-marathon run

Abstract

Context: Circulating miRNAs are potential biomarkers that can be important molecules driving cell-to-cell communication.

Objective: To investigate circulating muscle-specific miRNAs in recreational athletes.

Materials and methods: Three miRNAs from whole plasma before and after a half-marathon were analyzed by qPCR.

Results: MiR-1, ?133a, and ?206 significantly increased after the race.

Discussion: Increased levels of miRNAs after exercise point to potential biomarkers and to the possibility of being functional players following endurance training.

Conclusion: These miRNAs are potential biomarkers of muscle damage or adaptation to exercise.     

Dynamic Adipocyte Phosphoproteome Reveals that Akt Directly Regulates mTORC2

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Hydrogen sulphide inhibits cardiomyocyte hypertrophy by up-regulating miR-133a

Hydrogen sulphide (H₂S) has been shown to play a crucial role in cardiovascular physiology and disease. However, there is no information about the possible role of H₂S in cardiomyocyte hypertrophy (CH). Our results showed that pretreatment with NaHS, an H₂S donor, significantly reduced [³H]-leucine incorporation, cell surface area, mRNA expression of brain natriuretic peptide (BNP), intracellular reactive oxygen species (ROS), miR-21 and increased atrial natriuretic peptide (ANP) and miR-133a expression in hypertrophic cardiomyocytes. Anti-miR133a inhibitor transfection partly reduced the anti-hypertrophic effect of NaHS. In conclusion, H₂S is a direct inhibitor of CH; it acts by increasing miR-133a and inhibiting the increase in intracellular ROS.     

miR-181a Regulates Inflammation Responses in Monocytes and Macrophages

miR-181a has been presumed to target the 3′-untranslated regions (3′-UTR) of IL1a based on software predictions. miR-181a and IL1a have opposite expression levels in monocytes and macrophages in the inflammatory state. This led us to suspect that mir-181a has an important function in regulating inflammatory response by targeting IL1a. Fluorescence reporter assays showed that miR-181a effectively binds to the 3′-UTR of IL1a. The anti-inflammatory functions of miR-181a were investigated in lipopolysaccharides (LPS)-induced Raw264.7 and phorbol 12-myristate 13-acetate (PMA)/LPS-induced THP-1 cells. We found that miR-181a mimics significantly lowered IL1a expression levels in these cells and, interestingly, miR-181a inhibitors reversed this decrease. In addition, miR-181a mimics significantly inhibited increase in the levels of inflammatory factors (IL1b, IL6, and TNFa) in these cells. Furthermore, miR-181a mimics and inhibitors decreased and increased, respectively, production of reactive oxygen species in PMA/LPS-induced THP-1 cells. These results indicate that miR-181a regulates inflammatory responses by directly targeting the 3′-UTR of IL1a and down-regulating IL1a levels. Interestingly, we found that miR-181a inhibited production of inflammatory factors even in IL1a-induced THP-1 cells, suggesting that the anti-inflammatory effects of miR-181a possibly involves other targets in addition to IL1a. Thus, we provide the first evidence for anti-inflammatory effects of miR-181a mediated at least in part by down-regulating IL1a.     

The Aminosteroid Derivative RM-133 Shows In Vitro and In Vivo Antitumor Activity in Human Ovarian and Pancreatic Cancers

Ovarian and pancreatic cancers are two of the most aggressive and lethal cancers, whose management faces only limited therapeutic options. Typically, these tumors spread insidiously accompanied first with atypical symptoms, and usually shift to a drug resistance phenotype with the current pharmaceutical armamentarium. Thus, the development of new drugs acting via a different mechanism of action represents a clear priority. Herein, we are reporting for the first time that the aminosteroid derivative RM-133, developed in our laboratory, displays promising activity on two models of aggressive cancers, namely ovarian (OVCAR-3) and pancreatic (PANC-1) cancers. The IC₅₀ value of RM-133 was 0.8 μM and 0.3 μM for OVCAR-3 and PANC-1 cell lines in culture, respectively. Based on pharmacokinetic studies on RM-133 using 11 different vehicles, we selected two main vehicles: aqueous 0.4% methylcellulose:ethanol (92:8) and sunflower oil:ethanol (92:8) for in vivo studies. Using subcutaneous injection of RM-133 with the methylcellulose-based vehicle, growth of PANC-1 tumors xenografted to nude mice was inhibited by 63%. Quite interestingly, RM-133 injected subcutaneously with the methylcellulose-based or sunflower-based vehicles reduced OVCAR-3 xenograft growth by 122% and 100%, respectively. After the end of RM-133 treatment using the methylcellulose-based vehicle, OVCAR-3 tumor growth inhibition was maintained for ≥ 1 week. RM-133 was also well tolerated in the whole animal, no apparent sign of toxicity having been detected in the xenograft studies.     

Osteoblast Menin Regulates Bone Mass in Vivo

Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1^f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1^f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance.     

Reversine promotes browning of white adipocytes by suppressing miR-133a

Brown adipocytes are characterized by a high number of uncoupling protein 1 (UCP1)-positive mitochondrial content and increased thermogenic capacity. As UCP1-enriched cells can consume lipids by generating heat, browning of white adipocytes is now highlighted as a promising approach for the prevention of obesity and obesity-associated metabolic diseases. Upon cold exposure or β-adrenergic stimuli, downregulation of microRNA-133 (miR-133) elevates the expression levels of PR domain containing 16 (Prdm16), which has been shown to be a brown adipose determination factor, in brown adipose tissue and subcutaneous white adipose tissues (WAT). Here, we show that treatment of reversine to white adipocytes induces browning via suppression of miR-133a. Reversine treatment promoted the expression of brown adipocyte marker genes, such as Prdm16 and UCP1, increasing the mitochondrial content, while decreasing the levels of miR-133a and white adipocyte marker genes. Ectopic expression of miR-133a mimic reversed the browning effects of the reversine treatment. Moreover, intraperitoneal administration of reversine in mice upregulated thermogenesis and resulted in resistance to high-fat diet-mediated weight gain as well as browning of subcutaneous and epididymal WAT. Taken together, we found a novel way to promote browning of white adipocytes through downregulation of miR-133a followed by activation of Prdm16, with a synthetic chemical, reversine.     

Neogenin Regulates Skeletal Myofiber Size and Focal Adhesion Kinase and Extracellular Signal-regulated Kinase Activities In Vivo and In Vitro

A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.