Rodent Adipocytes Research Articles

Possibility of uncoupling protein 1 expression in bovine fast-twitch muscle fibers.

Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis in brown/beige adipocytes in humans and rodents. Previously, we showed unexpected expression of UCP1 in bovine skeletal muscles. Here we evaluated Ucp1 mRNA levels in the muscle tissue of Japanese Black steers. Expression of Ucp1 was higher in 30-month-old cattle than in 26-month-old cattle. Levels of myosin heavy chain (Myh)1, an MYH predominantly expressed in fast-twitch muscles, were also significantly higher in cattle aged 30 months. A similar tendency was observed in the expression of other Myhs that are highly expressed in fast-twitch muscles, Myh2 and Myh4. Ucp1 expression was positively correlated with expression of Myh1, Myh2, and Myh4. Our results indicate the possibility of Ucp1 expression in fast-twitch muscle fibers.

P300 or CBP is required for insulin-stimulated glucose uptake in skeletal muscle and adipocytes.

While current thinking posits that insulin signaling to glucose transporter 4 (GLUT4) exocytic translocation and glucose uptake in skeletal muscle and adipocytes is controlled by phosphorylation-based signaling, many proteins in this pathway are acetylated on lysine residues. However, the importance of acetylation and lysine acetyltransferases to insulin-stimulated glucose uptake is incompletely defined. Here, we demonstrate that combined loss of the acetyltransferases E1A binding protein p300 (p300) and cAMP response element binding protein binding protein (CBP) in mouse skeletal muscle caused a complete loss of insulin-stimulated glucose uptake. Similarly, brief (i.e., 1 hour) pharmacological inhibition of p300/CBP acetyltransferase activity recapitulated this phenotype in human and rodent myotubes, 3T3-L1 adipocytes, and mouse muscle. Mechanistically, these effects were due to p300/CBP-mediated regulation of GLUT4 exocytic translocation and occurred downstream of Akt signaling. Taken together, we highlight a fundamental role for acetylation and p300/CBP in the direct regulation of insulin-stimulated glucose transport in skeletal muscle and adipocytes.

Adropin Slightly Modulates Lipolysis, Lipogenesis and Expression of Adipokines but Not Glucose Uptake in Rodent Adipocytes.

Adropin is a peptide hormone which modulates energy homeostasis and metabolism. In animals with diet-induced obesity, adropin attenuates adiposity and improves lipid and glucose homeostasis. Adropin promotes the proliferation of rodent white preadipocytes and suppresses their differentiation into adipocytes. By contrast, the effects of adropin on mature white adipocytes are unknown. Therefore, we aimed to evaluate the effects of adropin on lipolysis, lipogenesis and glucose uptake in white rodent adipocytes. We assessed the effects of adropin on the mRNA expression of adiponectin, resistin and visfatin. White preadipocytes were isolated from male Wistar rats. Differentiated 3T3-L1 cells were used as a surrogate model of white adipocytes. Lipolysis was measured by the evaluation of glycerol and free fatty acid secretion using colorimetric kits. The effects of adropin on lipogenesis and glucose uptake were measured using radioactive-labelled glucose. The expression of adipokine mRNA was studied using real-time PCR. Our results show that adropin slightly promotes lipolysis in rat adipocytes and 3T3-L1 cells. Adropin suppresses lipogenesis in rat adipocytes without influencing glucose uptake. In addition, adropin stimulates adiponectin mRNA expression and suppresses the expression of resistin and visfatin. These results indicate that adropin may be involved in controlling lipid metabolism and adipokine expression in white rodent adipocytes.

Cardiovascular and Metabolic Crosstalk in the Brain: Leptin and Resistin.

Leptin and resistin are cytokines whose plasma levels correlate with adiposity. Leptin is a hormone synthesised and released from adipocytes and can be transported into the brain. Resistin is produced in adipocytes in rodents and in macrophages in humans, particularly macrophages that have infiltrated adipose tissue. Both hormones can act within the brain to influence sympathetic nerve activity. Leptin appears to have a generalised sympatho-excitatory actions whilst resistin appears to increase sympathetic nerve activity affecting the cardiovascular system but inhibits sympathetic nerve activity to brown adipose tissue, which contrasts with leptin. Since both hormones can be elevated in conditions of metabolic dysfunction, interactions/crosstalk between these two hormones in the brain is a real possibility. This review describes the current knowledge regarding such crosstalk within the central nervous system. The evidence suggests that with respect to sympathetic nerve activity, crosstalk between leptin and resistin can elicit enhanced sympatho-excitatory responses to the kidneys. In contrast, with respect to food intake, resistin has weaker effects, but in regard to insulin secretion and thermogenesis, leptin and resistin have opposing actions. Thus, in conditions in which there is increased resistin and leptin levels, the result of crosstalk in the central nervous system could contribute to worse cardiovascular and metabolic complications.

Regulatory expression of uncoupling protein 1 and its related genes by endogenous activity of the transforming growth factor-β family in bovine myogenic cells.

Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis, with restricted expression in brown/beige adipocytes in humans and rodents. We have previously shown an unexpected expression of UCP1 in bovine skeletal muscles. This study evaluated factors affecting Ucp1 gene expression in cultured bovine myogenic cells. Myosatellite cells, which were isolated from the bovine musculus longissimus cervicis, were induced to differentiate into myotubes in the presence of 2% horse serum. Previous studies using murine brown/beige adipocytes revealed that Ucp1 expression levels are directly increased by forskolin and all-trans retinoic acid (RA). The transforming growth factor-β (TGF-β)/activin pathway negatively regulated Ucp1 expression, whereas activation of the bone morphogenetic protein (BMP) pathway indirectly increases Ucp1 expression through the stimulation of brown/beige adipogenesis. Neither forskolin nor RA significantly affected Ucp1 mRNA levels in bovine myogenic cells. A-83-01, an inhibitor of the TGF-β/activin pathway, stimulated myogenesis in these cells. A-83-01 significantly increased the expression of some brown fat signature genes such as Pgc-1α, Cox7a1, and Dio2, with a quantitative but not significant increase in the expression of Ucp1. Treatment with LDN-193189, an inhibitor of the BMP pathway, did not affect the differentiation of bovine myosatellite cells. Rather, LDN-193189 increased Ucp1 mRNA levels without modulating the levels of other brown/beige adipocyte-related genes. The current results indicate that the regulation of Ucp1 expression in bovine myogenic cells is distinct from that in murine brown/beige adipocytes, which has been more intensely characterized. SIGNIFICANCE OF THE STUDY: We previously reported unexpected expression of Ucp1 in bovine muscle tissues; Ucp1 expression has been known to be detected predominantly in brown/beige adipocytes. This study examined regulatory expression of bovine Ucp1 in myogenic cells. Consistent with the changes in expression levels of brown/beige adipocyte-selective genes, Ucp1 expression tended to be increased by inhibition of endogenous TGF-β activity. In contrast, inhibition of endogenous BMP significantly increased Ucp1 expression without affecting brown/beige adipocyte-selective gene expression. The current results indicate that regulatory expression of Ucp1 in bovine myogenic cells is distinct from that in murine brown/beige adipocytes that is more intensely characterized.

Opipramol Inhibits Lipolysis in Human Adipocytes without Altering Glucose Uptake and Differently from Antipsychotic and Antidepressant Drugs with Adverse Effects on Body Weight Control.

Treatment with several antipsychotic drugs exhibits a tendency to induce weight gain and diabetic complications. The proposed mechanisms by which the atypical antipsychotic drug olanzapine increases body weight include central dysregulations leading to hyperphagia and direct peripheral impairment of fat cell lipolysis. Several investigations have reproduced in vitro direct actions of antipsychotics on rodent adipocytes, cultured preadipocytes, or human adipose tissue-derived stem cells. However, to our knowledge, no such direct action has been described in human mature adipocytes. The aim of the present study was to compare in human adipocytes the putative direct alterations of lipolysis by antipsychotics (haloperidol, olanzapine, ziprazidone, risperidone), antidepressants (pargyline, phenelzine), or anxiolytics (opipramol). Lipolytic responses to the tested drugs, and to recognized lipolytic (e.g., isoprenaline) or antilipolytic agents (e.g., insulin) were determined, together with glucose transport and amine oxidase activities in abdominal subcutaneous adipocytes from individuals undergoing plastic surgery. None of the tested drugs were lipolytic. Surprisingly, only opipramol exhibited substantial antilipolytic properties in the micromolar to millimolar range. An opipramol antilipolytic effect was evident against isoprenaline-, forskolin-, or atrial natriuretic peptide-stimulated lipolysis. Opipramol did not impair insulin activation of glucose transport but inhibited monoamine oxidase (MAO) activity to the same extent as antidepressants recognized as MAO inhibitors (pargyline, harmine, or phenelzine), whereas antipsychotics were inefficient. Considering its unique properties, opipramol, which is not associated with weight gain in treated patients, is a good candidate for drug repurposing because it limits exaggerated lipolysis, prevents hydrogen peroxide release by amine oxidases in adipocytes, and is thereby of potential use to limit lipotoxicity and oxidative stress, two deleterious complications of diabetes and obesity.

Primary Adipocytes as a Model for Insulin Sensitivity.

Obesity and its comorbidity insulin resistance lead to the development of chronic metabolic diseases, such as impaired fasted blood glucose and type 2 diabetes. Adipose tissue plays an important role in whole-body glucose homeostasis, particularly in obese individuals; therefore, many in vivo models of type 2 diabetes are obese, such as Lepob/ob and Leprdb/db mice or ZDF rats. Primary adipocytes therefore represent an attractive in vitro model to study insulin-mediated glucose uptake to investigate the mechanisms of insulin resistance and explore the potential insulin-sensitizing properties of new antidiabetic drugs.Primary adipocytes are isolated by collagenase digestion of adipose tissue, Glucose transport is evaluated by the measurement of intracellular uptake of a tracer (D-[U14C] glucose). The uptake of [U-14 C] glucose reflects directly glucose transport.In this chapter, we will describe the protocol for the isolation of primary rodent adipocytes and the measurement of basal and insulin-stimulated glucose uptake.

Cellular Origins of Beige Fat Cells Revisited.

Activated beige adipocytes have therapeutic potential due to their ability to improve glucose and lipid homeostasis. To date, the origin of beige adipocytes remains enigmatic. Whether beige cells arise through de novo differentiation from resident precursors or through reprogramming of mature white adipocytes has been a topic of intense discussion. Here, we offer our perspective on the natural origin of beige adipocytes in mice. In particular, we revisit recent lineage-tracing studies that shed light on this issue and offer new insight into how environmental housing temperatures early in life influence the mode of beige adipocyte biogenesis upon cold exposure later in life. We suggest a unified model in which beige adipocytes (UCP1+ multilocular cells) in rodents initially arise predominantly from progenitors (i.e., de novo beige adipogenesis) upon the first exposure to cold temperatures and then interconvert between "dormant beige" and "active beige" phenotypes (i.e., beige cell activation) upon subsequent changes in environmental temperature. Importantly, we highlight experimental considerations needed to visualize de novo adipogenesis versus beige cell activation in mice. A precise understanding of the cellular origins of beige adipocytes emanating in response to physiological and pharmacological stimuli may better inform therapeutic strategies to recruit beige adipocytes in vivo.

Reduced SIRT1 and SIRT2 expression promotes adipogenesis of human visceral adipose stem cells and associates with accumulation of visceral fat in human obesity.

The histone deacetylases SIRT1 and SIRT2 have been shown to be involved in the differentiation of rodent adipocyte precursors. In light of the differences in gene expression and metabolic function of visceral (V) and subcutaneous (S) adipose tissue (AT) and their resident cells, the aim of this study was to investigate the role of SIRT1 and SIRT2 in the differentiation of adipose stem cells (ASCs) isolated from SAT and VAT biopsies of nondiabetic obese and nonobese individuals. Human ASCs were isolated from paired SAT and VAT biopsies obtained from 83 nonobese and 92 obese subjects and were differentiated in vitro. Adipogenesis was evaluated by analyzing the lipid deposition using an image processing software, and gene expression by RT-qPCR. SIRT1 and SIRT2 protein expression was modified by using recombinant adenoviral vectors. Visceral but not subcutaneous ASCs from obese subjects showed an intrinsic increase in both adipogenesis and lipid accumulation when compared with ASCs from nonobese subjects, and this was associated with reduced SIRT1 and SIRT2 mRNA and protein levels. Moreover, adipose tissue mRNA levels of SIRT1 and SIRT2 showed an inverse correlation with BMI in the visceral but not subcutaneous depot. Overexpression of SIRT1 or SIRT2 in visceral ASCs from obese subjects resulted in inhibition of adipocyte differentiation, whereas knockdown of SIRT1 or SIRT2 in visceral ASCs from nonobese subjects enhanced this process. Changes in SIRT1 or SIRT2 expression and adipocyte differentiation were paralleled by corresponding changes in PPARG, CEBPA, and other genes marking terminal adipocyte differentiation. SIRT1 and SIRT2 modulate the differentiation of human ASC. Reduced expression of SIRT1 and SIRT2 may enhance the differentiation capacity of visceral ASC in human obesity, fostering visceral adipose tissue expansion.

CAMP-MicroRNA-203-IFNγ network regulates subcutaneous white fat browning and glucose tolerance

ObjectiveBrown and beige adipocytes in humans and rodents are specialized to burn lipids for heat generation as a natural defense against cold and obesity, which is advantageous to metabolic homeostasis. MicroRNAs as another regulatory layer to regulate metabolic homeostasis attracted a lot of attentions. Our previous work revealed microRNA (miR)-203 as a brown adipocyte-enriched microRNA involved in brown adipocytes development. However, the potential role of miR-203 in adipose tissue metabolic homeostasis has not been determined in vivo. In this study, we investigate the potential role of miR-203 in subcutaneous white adipose tissue (sub-WAT) browning and metabolic homeostasis. MethodsWe investigated the relationship between miR-203 and energy homeostasis in adipose tissue from cold exposed, high fat diet (HFD) fed, ob/ob and db/db mice. The functions of miR-203 on sub-WAT browning were validated through miR-203 knockdown or overexpression. The miR-203 targeted signal pathway was screened by RNAseq analysis. Luciferase report assay, western blot, and qPCR were performed to establish the miR-203 related upstream and downstream signal pathway in vivo and in vitro. The functions of miR-203 on obesity and metabolic homeostasis were validated through GTT/ITT and western blot on high fat diet-induced obesity in C57 mice. ELISA was used to determine the concentration of IFN-γ. Flow cytometry analysis was performed to determine the infiltration of macrophages in adipose tissue. ResultsMiR-203 expression positively correlates with energy expenditure, and overexpression of miR-203 could enhance sub-WAT browning in normal diet (ND) condition. Mechanistically, the expression of miR-203 is activated by cAMP-dependent C/EBPβ up-regulation. Subsequently, miR-203 inhibits IFN-γ signal pathway activation by directly targeting Lyn, which is an activator of Jak1-Stat1. Moreover, the forced expression of miR-203 could improve insulin sensitivity and resist high fat diet-induced obesity by inhibiting IFN-γ. ConclusionsMicroRNA-203 (miR-203) promotes white adipose tissue browning in cold exposed mice and improves glucose tolerance in HFD fed mice by repressing IFN-γ. Since miR-203 is activated by cAMP-dependent C/EBPβ up-regulation and directly represses IFN-γ signal pathway, we declare that miR-203 acts as a messenger between cAMP signal pathway and IFN-γ signal pathway.

AMP-activated protein kinase complexes containing the β2 regulatory subunit are up-regulated during and contribute to adipogenesis.

AMP-activated protein kinase (AMPK) is a heterotrimer of α-catalytic and β- and γ-regulatory subunits that acts to regulate cellular and whole-body nutrient metabolism. The key role of AMPK in sensing energy status has led to significant interest in AMPK as a therapeutic target for dysfunctional metabolism in type 2 diabetes, insulin resistance and obesity. Despite the actions of AMPK in the liver and skeletal muscle being extensively studied, the role of AMPK in adipose tissue and adipocytes remains less well characterised. Small molecules that selectively influence AMPK heterotrimers containing specific AMPKβ subunit isoforms have been developed, including MT47-100, which selectively inhibits complexes containing AMPKβ2. AMPKβ1 and AMPKβ2 are the principal AMPKβ subunit isoforms in rodent liver and skeletal muscle, respectively, yet the contribution of specific AMPKβ isoforms to adipose tissue function, however, remains largely unknown. This study therefore sought to determine the contribution of AMPKβ subunit isoforms to adipocyte biology, focussing on adipogenesis. AMPKβ2 was the principal AMPKβ isoform in 3T3-L1 adipocytes, isolated rodent adipocytes and human subcutaneous adipose tissue, as assessed by the contribution to total cellular AMPK activity. Down-regulation of AMPKβ2 with siRNA inhibited lipid accumulation, cellular adiponectin levels and adiponectin secretion during 3T3-L1 adipogenesis, whereas down-regulation of AMPKβ1 had no effect. Incubation of 3T3-L1 cells with MT47-100 selectively inhibited AMPK complexes containing AMPKβ2 whilst simultaneously inhibiting cellular lipid accumulation as well as cellular levels and secretion of adiponectin. Taken together, these data indicate that increased expression of AMPKβ2 is an important feature of efficient adipogenesis.

PRDM16 Represses the Pig White Lipogenesis through Promoting Lipolysis Activity.

The positive regulatory domain containing 16 (PRDM16) gene is a dominant transcriptional regulator that favors the “browning” of white adipocytes in rodents. Since the “browning” of white fat is important in pig in terms of producing heat fighting against cold environment, avoiding obesity, and improving meat quality, understanding the critical role that PRDM16 gene played in pig adipose “browning” and energy metabolism is of great significance. However, the constitution of pig fat differs a lot from rodents and human as they do not have brown adipose tissue (BAT) even in the newborn piglets. In this study, we isolated porcine primary preadipocytes and investigated the function of PRDM16 during preadipocytes differentiation. Our results showed that overexpression of the PR domain of PRDM16 repressed the differentiation of porcine preadipocytes, indicated by oil red O staining and the deposition of the triglyceride. Overexpression of the PR domain significantly increased the level of lipolysis and mitochondrial oxidative capacity detected by Western blotting during differentiation. Furthermore, we purified the protein coded by the PR domain and demonstrated that this protein has the H3K9me1 methyltransferase activity. In conclusion, the PR domain of the porcine PRDM16 gene repressed the mature of the porcine preadipocytes by promoting its oxidative activity.

137-OR: The Selective Human Beta 3 Adrenergic Receptor Mirabegron Potently Activates Lipolysis in Human White Adipocytes

Human obesity is an increasing burden worldwide. Lipids stored as metabolic fuel in white adipose tissue (WAT) can be rapidly mobilized through lipolysis. In rodent adipocytes, activation of the β3-adrenergic receptors (β3-ARs) leads to lipolysis. The physiological role of the β3-AR in human adipocytes remains controversial due to its reported low expression and previous access only to partial β3-AR agonists. Mirabegron, an FDA-approved drug for overreactive bladder, is a highly-selective human β3-AR agonist. In clinical trials, mirabegron treatment led to activation of human brown adipose tissue (BAT) thermogenesis and increased plasma NEFA’s. Therefore, we investigated if mirabegron directly mediates lipolysis in human white adipocytes. We found similar gene expression of the three β-ARs in human immortalized and primary white adipocytes, with β2-AR being the most expressed, followed by β1-AR and β3-AR. Next, to assess the functionally of the β3-AR in human adipocytes, we established dose-response curves and found that mirabegron increased the release of glycerol, a lipolytic product, in the picomolar range. To identify the molecular mechanism controlling mirabegron-induced lipolysis, we assessed the regulation of key downstream lipolytic proteins. Mirabegron mediated lipolysis by rapidly phosphorylating hormone sensitive lipase (HSL), as well as the mitogen activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK). Moreover, acute inhibition of either the PKA or MAPK/ERK pathways decreased lipolysis. In summary, we identify a major role played by the β3-AR in the regulation of human WAT lipolysis. Mirabegron acts as a potent pro-lipolytic effector, signaling through the canonical adrenergic receptor pathway as well as the MAPK/ERK pathway. Due to the comparatively minimal effects previously reported of mirabegron on the cardiovascular system, mirabegron may be the optimal choice for targeted lipid mobilization in human WAT. Disclosure C. Cero: None. J.W. Johnson: None. A. O’Mara: None. J.D. Linderman: None. A.M. Cypess: None. Funding National Institutes of Health

Adrenoceptors in white, brown, and brite adipocytes.

Adrenoceptors play an important role in adipose tissue biology and physiology that includes regulating the synthesis and storage of triglycerides (lipogenesis), the breakdown of stored triglycerides (lipolysis), thermogenesis (heat production), glucose metabolism, and the secretion of adipocyte-derived hormones that can control whole-body energy homeostasis. These processes are regulated by the sympathetic nervous system through actions at different adrenoceptor subtypes expressed in adipose tissue depots. In this review, we have highlighted the role of adrenoceptor subtypes in white, brown, and brite adipocytes in both rodents and humans and have included detailed analysis of adrenoceptor expression in human adipose tissue and clonally derived adipocytes. We discuss important considerations when investigating adrenoceptor function in adipose tissue or adipocytes. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

Peptide/Receptor Evolution Explains the Lipolytic Function of the Neuropeptide TLQP-21

Functional diversity of peptide substrates and their cognate G-Protein Coupled Receptors (GPCR) are often the result of a long evolutionary process shaped by accumulation of mutations and natural selection. Even small changes in amino acid sequence can drastically alter their binding affinity and receptor activation. Since GPCR are often investigated as molecular targets for pharmacotherapies, it is fundamentally essential to experimentally compare peptide/receptor interaction when translating promising pre-clinical therapeutic efficacy into humans. Here, we focused on the role of the Complement 3a Receptor (C3aR1) activated by the VGF-derived neuropeptide TLQP-21 in rodent and human cell system, and used molecular modelling and evolutionary analysis to infer the exact mechanism of ligand/receptor interaction. After having characterized the mechanism of TLQP-21 potentiation of beta-Adrenergic Receptor (bARs)-induced lipolysis in rodent adipocytes, we used structural data-driven homology modeling and molecular dynamics simulation to identify the TLQP-21 binding motif and its corresponding C3aR1 binding site. We found that the mouse/rat TLQP-21 had enhanced binding affinity at the human C3aR1, when compared to the human TLQP-21. Similarly, the mouse/rat TLQP-21, but not the human TLQP-21, potentiate lipolysis in human adipocytes. This surprising finding led us to reconstruct the evolution of sequence motifs within the C3aR1 binding site and the TLQP-21 pharmacophore in the Murinae sub-family of rodents, to identify changes which explain the pharmacological efficacy by virtue of enhanced hydrophobicity, and which represent stark enhancement of function for a neuropeptide that is thus rendered a potent agonist at C3aR1. Since we further demonstrated that changes in gene expression of critical nodes in TLQP-21/C3aR1-mediated lipolysis are conserved in obesity for humans and mice, and that the rodent peptide is a potent agonist at the human C3aR1, our discovery can pave the way to investigate a peptide/receptor mechanism and to develop novel pharmacotherapies for obesity and other disease conditions in which C3aR1 has been implicated.

Orexin A but not orexin B regulates lipid metabolism and leptin secretion in isolated porcine adipocytes

It is well known that orexins are involved in the metabolism and endocrine function of rodent adipocytes, but there are no data on other animal species, including pigs. Therefore, in this study, we tested the hypothesis that orexin A (OxA) and orexin B (OxB) modulate the metabolism and endocrine functions of isolated porcine adipocytes and adipose tissue explants. Moreover, we characterized the possible mechanism of OxA action in porcine adipocytes. According to the results, both orexin receptor 1 and orexin receptor 2 were expressed in the porcine adipose tissue. We found that OxA suppressed the release of glycerol from porcine adipocytes both in the absence (basal lipolysis; P < 0.05) and in the presence (stimulated lipolysis; P < 0.05) of isoproterenol. Orexin A increased basal and insulin-stimulated glucose uptake (P < 0.05), as well as it enhanced the rate of glucose incorporation into lipids with insulin (stimulated lipogenesis; P < 0.01) or without insulin (basal; P < 0.05). We have also shown that OxA stimulated the mRNA expression of glucose transporter 4 (P < 0.05) and its translocation into the plasma membrane (P < 0.01). Moreover, OxA upregulated the mRNA expression of leptin in isolated porcine adipocytes (P < 0.05) and increased the secretion of leptin (P < 0.05). We have also demonstrated one of the possible mechanisms of OxA action in adipocytes. In the presence of extracellular-signal-regulated kinase 1 and 2 (ERK1/2) inhibitor, the effect of OxA was not detectable in porcine adipocytes, which indicates that this peptide increased cell viability via ERK1/2 pathway (P < 0.05). However, OxB did not show any effect on the metabolism and endocrine functions of porcine adipocytes. In summary, we have shown for the first time that OxA has a significant impact on the intensity of lipolysis, glucose uptake, lipogenesis, as well as on the expression and secretion of leptin. Therefore, we conclude that OxA but not OxB regulates lipid metabolism in porcine adipose tissue and that this regulation is partly mediated via ERK1/2 pathway. The action of orexins should be further explored to better understand their role in the regulation of adiposity in pigs.

Second messenger signaling mechanisms of the brown adipocyte thermogenic program: an integrative perspective.

β-adrenergic receptors (βARs) are well established for conveying the signal from catecholamines to adipocytes. Acting through the second messenger cyclic adenosine monophosphate (cAMP) they stimulate lipolysis and also increase the activity of brown adipocytes and the 'browning' of adipocytes within white fat depots (so-called 'brite' or 'beige' adipocytes). Brown adipose tissue mitochondria are enriched with uncoupling protein 1 (UCP1), which is a regulated proton channel that allows the dissipation of chemical energy in the form of heat. The discovery of functional brown adipocytes in humans and inducible brown-like ('beige' or 'brite') adipocytes in rodents have suggested that recruitment and activation of these thermogenic adipocytes could be a promising strategy to increase energy expenditure for obesity therapy. More recently, the cardiac natriuretic peptides and their second messenger cyclic guanosine monophosphate (cGMP) have gained attention as a parallel signaling pathway in adipocytes, with some unique features. In this review, we begin with some important historical work that touches upon the regulation of brown adipocyte development and physiology. We then provide a synopsis of some recent advances in the signaling cascades from β-adrenergic agonists and natriuretic peptides to drive thermogenic gene expression in the adipocytes and how these two pathways converge at a number of unexpected points. Finally, moving from the physiologic hormonal signaling, we discuss yet another level of control downstream of these signals: the growing appreciation of the emerging roles of non-coding RNAs as important regulators of brown adipocyte formation and function. In this review, we discuss new developments in our understanding of the signaling mechanisms and factors including new secreted proteins and novel non-coding RNAs that control the function as well as the plasticity of the brown/beige adipose tissue as it responds to the energy needs and environmental conditions of the organism.

Antiobesity Action of ACAM by Modulating the Dynamics of Cell Adhesion and Actin Polymerization in Adipocytes.

Coxsackie virus and adenovirus receptor-like membrane protein (CLMP) was identified as the tight junction-associated transmembrane protein of epithelial cells with homophilic binding activities. CLMP is also recognized as adipocyte adhesion molecule (ACAM), and it is upregulated in mature adipocytes in rodents and humans with obesity. Here, we present that aP2 promoter-driven ACAM transgenic mice are protected from obesity and diabetes with the prominent reduction of adipose tissue mass and smaller size of adipocytes. ACAM is abundantly expressed on plasma membrane of mature adipocytes and associated with formation of phalloidin-positive polymerized form of cortical actin (F-actin). By electron microscopy, the structure of zonula adherens with an intercellular space of ∼10-20 nm was observed with strict parallelism of the adjoining cell membranes over distances of 1-20 μm, where ACAM and γ-actin are abundantly expressed. The formation of zonula adherens may increase the mechanical strength, inhibit the adipocyte hypertrophy, and improve the insulin sensitivity.

Structural Basis for Nucleotide Hydrolysis by the Acid Sphingomyelinase-like Phosphodiesterase SMPDL3A

Sphingomyelin phosphodiesterase, acid-like 3A (SMPDL3A) is a member of a small family of proteins founded by the well characterized lysosomal enzyme, acid sphingomyelinase (ASMase). ASMase converts sphingomyelin into the signaling lipid, ceramide. It was recently discovered that, in contrast to ASMase, SMPDL3A is inactive against sphingomyelin and, surprisingly, can instead hydrolyze nucleoside diphosphates and triphosphates, which may play a role in purinergic signaling. As none of the ASMase-like proteins has been structurally characterized to date, the molecular basis for their substrate preferences is unknown. Here we report crystal structures of murine SMPDL3A, which represent the first structures of an ASMase-like protein. The catalytic domain consists of a central mixed β-sandwich surrounded by α-helices. Additionally, SMPDL3A possesses a unique C-terminal domain formed from a cluster of four α-helices that appears to distinguish this protein family from other phosphoesterases. We show that SMDPL3A is a di-zinc-dependent enzyme with an active site configuration that suggests a mechanism of phosphodiester hydrolysis by a metal-activated water molecule and protonation of the leaving group by a histidine residue. Co-crystal structures of SMPDL3A with AMP and α,β-methylene ADP (AMPCP) reveal that the substrate binding site accommodates nucleotides by establishing interactions with their base, sugar, and phosphate moieties, with the latter the major contributor to binding affinity. Our study provides the structural basis for SMPDL3A substrate specificity and sheds new light on the function of ASMase-like proteins.

Regulation of G0/G1 switch gene 2 (G0S2) expression in human adipose tissue

The G0/G1 switch gene 2 (G0S2) protein attenuated adipose triglyceride lipase (ATGL) activity and decreased lipolysis in rodent and human adipocytes. We hypothesized that G0S2 mRNA expression in human adipose tissue is influenced by depot, adipocyte size, body weight and caloric intake. Adipose tissue samples were obtained during abdominal surgery and by needle biopsy before and 3 h after an extended glucose load in lean subjects. G0S2 mRNA was 7× higher expressed in mature human adipocytes compared to the stromavascular fraction. Cell size inversely correlated with G0S2 mRNA expression in both, subcutaneous and omental adipose depots. G0S2 mRNA expression was 75% higher in subcutaneous compared to omental adipose tissue. Obesity was associated with lower G0S2 mRNA expression in subcutaneous adipose tissue. Acute glucose ingestion after an overnight fast did not significantly increase G0S2 expression in subcutaneous adipose tissue. In conclusion, differences in G0S2 expression may explain depot-specific and obesity-associated differences in lipolysis on the molecular level.