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Nicotinamide adenine dinucleotide ([NAD.sup.+]) acts as a redox cofactor for more than 200 enzymatic reactions and serves as a cosubstrate for the sirtuins, a family of [NAD.sup.+]-dependent deacetylases (1-3). Sirtuin [...], Nicotinamide N-methyltransferase (Nnmt) methylates nicotinamide, a form of vitamin B3, to produce [N.sup.1]-methylnicotinamide (MNAM). Nnmt has emerged as a metabolic regulator in adipocytes, but its role in the liver, the tissue with the strongest Nnmt expression, is not known. In spite of its overall high expression, here we find that hepatic expression of Nnmt is highly variable and correlates with multiple metabolic parameters in mice and humans. Further, we find that suppression of hepatic Nnmt expression in vivo alters glucose and cholesterol metabolism and that the metabolic effects of Nnmt in the liver are mediated by its product MNAM. Supplementation of high-fat diet with MNAM decreases serum and liver cholesterol and liver triglycerides levels in mice. Mechanistically, increasing Nnmt expression or MNAM levels stabilizes sirtuin 1 protein, an effect that is required for their metabolic benefits. In summary, we describe here a novel regulatory pathway for vitamin B3 that could provide a new opportunity for metabolic disease therapy.

Using suppression subtractive hybridization technique, we found that 2 novel genes (AEUG1 and AEUG3) were highly expressed in the adipocytes compared with preadipocytes. We then identified that these 2 genes were both angiotensin-converting enzyme 2 (ACE2). We applied 3'RACE (rapid amplification of cDNA end), 5'RACE, and PCR to obtain the full-length porcine ACE2 cDNA sequence. Because eicosanoids derived from PUFA are involved in regulating blood pressure, we hypothesized that PUFA can regulate the expression of the vasodilator ACE2. Preadipocytes from Landrace pigs were induced to differentiate for 4 d, then treated with 50 [micro]M of different PUFA, CLA, arachidonic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or stearic acid (18:0). Addition of EPA or 18:0 for 48 h did not change the ACE2 mRNA abundance, whereas the treatments of arachidonic acid, CLA, and DHA significantly decreased ACE2 mRNA abundance after 48 h (P [less than or equal to] 0.05). Treatment with PUFA did not change (P > 0.05) type I and type II angiotensin receptor mRNA abundance. To further understand how PUFA metabolites affect ACE2 mRNA expression, we inhibited individual enzymes that are involved in eicosanoid production. We found that 3 individual eicosanoid pathway enzyme inhibitors recovered the PUFA effect on the expression of ACE2, indicating these pathways are involved in mediating the PUFA function. In conclusion, we obtained the full-length porcine ACE2 cDNA sequence and found PUFA could downregulate the expression of ACE2 through its metabolites without changing the expression of their receptor in porcine adipocytes. Key words: amplification of complementary deoxyribonucleic acid end, angiotensin-converting enzyme, polyunsaturated fatty acid doi: 10.2527/jas.2010-2905

Visfatin is a visceral adipose tissue-specific adipocytokine that plays a positive role in attenuating insulin resistance by binding to the insulin receptor. Visfatin has been suggested to play a role in the regulation of lipid metabolism and inflammation; however, the mechanism remains unclear. We investigated the effects of visfatin on the regulation of gene expression in cultured porcine preadipocytes and differentiated adipocytes. In preadipocytes, the mRNA abundance of lipoprotein lipase and PPAR[gamma] were significantly increased by visfatin or insulin treatment after 8 d (all P < 0.05). In the presence of insulin, the mRNA abundance of adipocyte fatty acid-binding protein was 24.7-fold greater than in the untreated group (P < 0.05), whereas visfatin alone had no effect on adipocyte fatty acid-binding protein mRNA abundance. Adipocyte differentiation was induced by insulin treatment for 8 d. In differentiated porcine adipocytes, exposure to insulin or visfatin for 24 h increased (P < 0.05) fatty acid synthase mRNA abundance but had no effect on the expression of sterol regulatory element binding-protein 1c mRNA. We also found a 5.8-fold upregulation of IL-6 expression in porcine adipocytes after 24 h of treatment with visfatin (P < 0.05). These results demonstrated that visfatin upregulated lipoprotein lipase expression in preadipocytes, potentially facilitating lipid uptake, and increased the gene expression of fatty acid synthase in differentiated adipocytes to potentially enhance lipogenic activity. Furthermore, visfatin can upregulate IL-6 expression in differentiated porcine adipocytes. The information presented in this study provides insights into the roles of visfatin in lipid metabolism in pigs. Key words: adipocyte, lipid metabolism, visfatin doi: 10.2527/jas.2010-2799

Introduction Obesity and type 2 diabetes are associated with elevated levels of plasma free fatty acids, which is thought to result from dysregulated lipolysis in adipocytes (Bergman and Ader 2000). [...], Interleukin (IL)-6, a proinflammatory cytokine, stimulates adipocyte lipolysis and induces insulin resistance in obese and diabetic subjects. However, the effects of the anti-diabetic drug rosiglitazone on IL-6-stimulated lipolysis and the underlying molecular mechanism are largely unknown. In this study, we demonstrated that rosiglitazone suppressed IL-6-stimulated lipolysis in differentiated porcine adipocytes by inactivation of extracellular signal-related kinase (ERK). Meanwhile, rosiglitazone enhanced the lipolysis response of adipocytes to isoprenaline. In addition, rosiglitazone significantly reversed IL-6-induced down-regulation of several genes such as perilipin A, peroxisome proliferators activated receptor gamma (PPARγ), and fatty acid synthetase, as well as the up-regulation of IL-6 mRNA. However, mRNA expression of PPARγ coactivator-1 alpha (PCG-1a) was enhanced by rosiglitazone in IL-6-stimulated adipocytes. These results indicate that rosiglitazone suppresses IL-6-stimulated lipolysis in porcine adipocytes through multiple molecular mechanisms. Key words: rosiglitazone, IL-6, adipocytes, lipolysis. L'interleukine (IL)-6, une cytokine proinflammatoire, stimule la lipolyse chez les adipocytes et induit la resistance a l'insuline chez les sujets obeses et diabetiques. Cependant, les effets du medicament anti-diabetique rosiglitazone sur la lipolyse stimuleee par l'IL-6 et les mecanismes moleculaires sous-jacents sont pratiquement inconnus. Dans cette etude, nous avons demontre que le rosiglitazone supprimait la lipolyse stimulee par l'IL-6 chez les adipocytes porcins differencies suite a l'inactivation de la kinase ERK (extracellular signal-related kinase). En meme temps, le rosiglitazone augmentait la lipolyse des adipocytes en reponse a l'isoprenaline. De plus, le rosiglitazone renversait significativement la diminution de la transcription induite par l'IL-6 de plusieurs genes, notamment la perilipine A, le PPAR-g (peroxysome proliferators activated receptor gamma) et la synthase d'acides gras, ainsi que la regulation a la hausse de l'ARNm de l'IL-6. Cependant, l'expression de l'ARNm du coactivateur de PPAR-g (PCG-la)etait augmentee par le rosiglitazone dans les adipocytes stimules par l'IL-6. En somme, ces resultats indiquent que le rosiglitazone supprime la lipolyse stimutee par l'IL-6 dans les adipocytes porcins par differents mecanismes. Mots-cles: rosiglitazone, IL-6, adipocytes, lipolyse. [Traduit par la Redaction]

In this study, we explored the roles of microRNAs in adipocyte differentiation and metabolism. We first knocked down Argonaute2 (Ago2), a key enzyme in the processing of micro-RNAs (miRNAs), to investigate a potential role for miRNAs in adipocyte differentiation and/or metabolism. Although we did not observe dramatic differences in adipogenesis between Ago2 knock-down and control 3T3-L1 cells, incorporation of [[sup.14]C]glucose or acetate into triacylglycerol, and steady-state levels of triacyglycerol were all reduced, suggesting a role for miRNAs in adipocyte metabolism. To study roles of specific miRNAs in adipocyte biology, we screened for miRNAs that are differentially expressed between preadipocytes and adipocytes for the 3T3-L1 and ST2 cell lines. Distinct subsets of miRNAs decline or increase during adipocyte conversion, whereas most miRNAs are not regulated. One locus encoding two miRNAs, 378/378', contained within the intron of PGC-1[beta] is highly induced during adipogenesis. When overexpressed in ST2 mesenchymal precursor cells, [miRNA378/378.sup.*] increases the size of lipid droplets and incorporation of [[sup.14]C]acetate into triacyl-glycerol. Although protein and mRNA expression levels of C/EBP[alpha], C/EBP[beta], C/EBP[delta], and PPAR[gamma]1 are unchanged, microarray and quantitative RT-PCR analyses indicate that a set of lipogenic genes are upregulated, perhaps due to increased expression of PPAR[gamma]2. Knockdown of miRNA378 and/or [miRNA378.sup.*] decreases accumulation of triacylglycerol. Interestingly, we made the unexpected finding that [miRNA378/378.sup.*] specifically increases transcriptional activity of C/EBP[alpha] and C/EBP[beta] on adipocyte gene promoters. adipogenesis; PPAR[gamma], coactivator-l[beta]; micro-RNA doi: 10.1152/ajpendo.00179.2010.

Obesity-associated inflammation is characterized by recruitment of macrophages (M[phi]) into white adipose tissue (WAT) and production of inflammatory cytokines, leading to the development of insulin resistance. The xanthones, [alpha]- and [gamma]-mangostin (MG), are major bioactive compounds found in mangosteen that are reported to have antiinflammatory and antioxidant properties. Thus, we examined the efficacy of MG to prevent lipopolysaccharide (LPS)-mediated inflammation in human M[phi] (differentiated U937 cells) and cross-talk with primary cultures of newly differentiated human adipocytes. We found that [alpha]- and [gamma]-MG attenuated LPS-induced expression of inflammatory genes, including tumor necrosis factor-[alpha], interleukin-6, and interferon [gamma]-inducible protein-10 in a dose-dependent manner in M[phi]. We also found that [alpha]- and [gamma]-MG attenuated LPS-activated mitogen-activated protein kinases (MAPK) and activator protein (AP)-1, but only [gamma]-MG reduced nuclear factor-[kappa]B (NF-[kappa]B). In addition, [alpha]- and [gamma]-MG attenuated LPS suppression of PPAR[gamma]/gene expression in a dose-dependent manner. Notably, the ability of M[phi]-conditioned media to cause inflammation and insulin resistance in primary cultures of human adipocytes was attenuated by pretreating M[phi] with [gamma]-MG. Taken together, these data demonstrate that MG attenuates LPS-mediated inflammation in M[phi] and insulin resistance in adipocytes, possibly by preventing the activation of MAPK, NF-[kappa]B, and AP-1, which are central to inflammatory cytokine production in WAT. doi: 10.3945/jn.109.120022

Glucose entry into cells is mediated by a family of facilitative transporter proteins (GLUTs). In mammals, GLUT4 is expressed in insulin-sensitive tissues and is responsible for the postprandial uptake of glucose. In fish, GLUT4 also mediates insulin-regulated glucose entry into cells but differs from mammalian GLUT4 in its affinity for glucose and in protein motifs known to be important for the traffic of GLUT4. In this study, we have characterized the intracellular and plasma membrane (PM) traffic of two orthologs of GLUT4 in fish, trout (btGLUT4) and salmon (okGLUT4), that do not share the amino terminal FQQI targeting motif of mammalian GLUT4. btGLUT4 (FQHL) and, to a lesser extent, okGLUT4 (FQQL) showed higher basal PM levels, faster traffic to the PM after biosynthesis, and earlier acquisition of insulin responsiveness than rat GLUT4. Furthermore, btGLUT4 showed a similar profile of internalization than rat GLUT4. Expression of the dominant-interfering AS160-4P mutant caused a significant decrease in the insulin-induced PM levels of okGLUT4 and rat GLUT4 and, to a lesser extent, of btGLUT4, suggesting that btGLUT4 has reduced retention into the IRC. Contrary to rat GLUT4 and okGLUT4, the presence of btGLUT4 at the PM under insulinstimulated conditions was not affected by coexpression of a dominant-interfering GGA mutant. These data suggest that fish GLUT4 follow a different trafficking pathway to the PM compared with rat GLUT4 that seems to be relatively independent of GGA. These results indicate that the regulated trafficking characteristics of GLUT4 have been modified during evolution from fish to mammals. Golgi-localized [gamma]-ear-containing Arf-binding protein; trout; salmon; glucose transport; glucose transporter 4; 3T3-L1 cells doi: 10.1152/ajpendo.00547.2009

Wang M, Wang JJ, Li J, Park K, Qian X, Ma J, Zhang SX. Pigment epithelium-derived factor suppresses adipogenesis via inhibition of the MAPK/ERK pathway in 3T3-L1 preadipocytes. Am J Physiol Endocrinol Metab 297: E1378-E1387, 2009. First published October 6, 2009:doi: 10.1152/ajpendo.00252.2009.--We previously reported that circulating levels of pigment epithelium-derived factor (PEDF), a newly identified adipokine, are increased in patients with type 2 diabetes, correlating with body mass index. However, the role of PEDF in adipogenesis remains elusive. In the present study, we have investigated the effects and mechanisms of PEDF on adipocyte differentiation in 3T3-L1 preadipocytes. Differentiation of 3T3-L1 preadipocytes was induced in the presence or absence of human recombinant PEDF protein. The effects of PEDF on adipogenic gene expression, mitotic clonal expansion (MCE), and MAPK activation were investigated. Physiological concentrations of human PEDF protein inhibited adipocyte differentiation, evidenced by decreased lipid accumulation, downregulation of adipocyte markers, and inhibition of master adipogenic transcription factors such as C/EBP-[alpha] and PPAR[gamma] The antiadipogenic effects of PEDF were observed only when PEDF was added to the cells on day 0 but not on day 3 during differentiation, suggesting that PEDF targets some early adipogenic events. Similarly, overexpression of PEDF by adenovirus attenuated adipocyte differentiation. Further studies revealed that PEDF, or U-0126, a specific MAPK/ERK inhibitor, sequentially inhibited the early activation of ERK and MCE. Moreover, PEDF attenuated expression and the phosphorylation of C/EBP-[beta] at [Thr.sup.188], an essential step for transcriptional activation of C/EBP-[beta]. In addition, PEDF expression was decreased significantly in the first 24 h during adipocyte differentiation, suggesting that downregulation of PEDF may be essential for the initiation of MCE and adipogenesis. We conclude that PEDF inhibits adipogenesis in 3T3-L1 preadipocytes partially because of inhibition of the MAPK/ERK signaling pathway and MCE. mitogen-activated protein kinase; extracellular signal-regulated kinase doi: 10.1152/ajpendo.00252.2009

Increases in key components of adipogenesis and lipolysis pathways correlate at the population-averaged level during adipogenesis. However, differentiating preadipocytes are highly heterogeneous in cellular and lipid droplet (LD) morphologies, and the degree to which individual cells follow population-averaged trends is unclear. In this study, we analyze the molecular heterogeneity of differentiating 3T3-L1 preadipocytes using immuno-fluorescence microscopy. Unexpectedly, we only observe a small percentage of cells with high simultaneous expression of markers for adipogenesis (peroxisome proliferator-activated receptor [gamma] [PPAR[gamma]], CCAAT/enhancer-binding protein [alpha], and adiponectin) and lipid accumulation (hormone-sensitive lipase, perilipin A, and LDs). Instead, we identify subpopulations of cells with negatively correlated expressions of these readouts. Acute perturbation of adipocyte differentiation with PPAR[gamma], agonists, forskolin, and fatty acids induced subpopulation-specific effects, including redistribution of the percentage of cells in observed subpopulations and differential expression levels of PPAR[gamma]. Collectively, our results suggested that heterogeneity observed during 3T3-L1 adipogenesis reflects a dynamic mixture of subpopulations with distinct physiological states. doi/10.1083/jcb.200904140

An approach is described for measuring human subcutaneous fat thickness automatically by using ultrasound radio frequency (RF) signals. High correlations are observed between the manual and automatic ultrasound measurements and between the skinfold caliper and automatic ultrasound measurements.

Obesity is associated with impaired functional hyperemic response. We have shown that ATP-sensitive potassium ([K.sub.ATP]) channels are important in mediating functional vasodilation. Adipocyte-derived factors (ADFs) can alter vascular tone via opening [K.sub.ATP] channels. We hypothesize that, in an animal model of obesity, ADFs will decrease basal arteriolar tone by opening [K.sub.ATP] channels, resulting in an attenuated functional vasodilation. We used wild-type (WT) mice and [ob.sup.-]/[ob.sup.-] mice (ob) to test this hypothesis. The spinotrapezius muscle was prepared for the microcirculatory observation of arcade arterioles, and we measured the vasodilatory responses to muscle stimulation. The basal arteriolar diameter was larger in ob mice compared with WT mice. The [K.sub.ATP] channel inhibitor glibenclamide (10 [micro]M) decreased arteriolar diameter in ob mice with no effect in WT mice. The increase in arteriolar diameter induced by muscle stimulation was attenuated in ob mice compared with WT mice. To determine the mechanisms for the opening of [K.sub.ATP] channels, fat was collected from the ob mice, subcutaneous fat from around the spinotrapezius muscle (OBSF) or visceral fat (OBVF) and was incubated in physiological saline solution (PSS). The vasodilatory responses to the fat-conditioned PSS were determined in WT mice. Treatment with OBSF- or OBVF -conditioned PSS increased the arteriolar diameters in WT mice, a dilation that was inhibited by glibenclamide. The absolute diameters induced by muscle stimulation were not altered by the fat-conditioned PSS. These results suggest that, in ob mice, local ADFs reduce the functional vasodilatory capability via opening [K.sub.ATP] channels. obesity; cytokines; vasodilation; adipose tissue; and potassium channels

Salt-inducible kinase 2 (SIK2) is expressed abundantly in adipose tissues and represses cAMP-response element-binding protein (CREB)-mediated gene expression by phosphorylating the coactivator transducer of regulated CREB activity (TORC2). Phosphorylation at [Ser.sup.587] of SIK2 diminishes its TORC2 phosphorylation activity. In 3T3-LI white adipocytes, SIK2 downregulates lipogenic gene in response to nutritional stresses. To investigate the impact of SIK2 on the function of brown adipose tissue (BAT), we used T37i brown adipocytes, mice with diet-induced obesity, and SIK2 mutant (S587A) transgenic mice. When T37i adipocytes were treated with insulin, the levels of peroxisome proliferator-activated receptor-coactivator-l[alpha] (PGC-1[alpha]) and uncoupling protein-1 (UCP-1) mRNA were increased, and the induction was inhibited by overexpression of SIK2 (S587A) mutant or dominant-negative CREB. Insulin enhanced SIK2 phosphorylation at [Ser.sup.587], which was accompanied by decrease in phospho-TORC2. Similarly, the decrease in the level of SIK2 phosphorylation at [Ser.sup.587] was observed in the BAT of mice with diet-induced obesity, which was negatively correlated with TORC2 phosphorylation. To confirm the negative correlation between SIK2 phosphorylation at [Ser.sup.587] and TORC2 phosphorylation in BAT, SIK2 mutant (S587A) was overexpressed in adipose tissues by using the adipocyte fatty acid-binding protein 2 promoter. The expression of recombinant SIK2 (S587A) was restricted to BAT, and the levels of phospho-TORC2 were elevated in BAT of transgenic mice. Male transgenic mice developed high-fat diet-induced obesity, and their BAT expressed low levels of PGC-1[alpha] and UCP-1 mRNA, suggesting that SIK2-TORC2 cascade may be important for the regulation of PGC-1[alpha] and UCP-1 gene expression in insulin signaling in BAT. salt-inducible kinase 2; peroxisome proliferator-activated receptorcoactivator-l[alpha]; uncoupling protein-l; brown adipocyte; adenosine 5',3'-cyclic monophosphate-response element-binding protein

Whereas adipose tissue possesses a local renin-angiotensin system, the synthesis and regulated release of renin has not been addressed. To that end, we utilized differentiating 3T3-L1 cells and analyzed renin expression and secretion. Renin mRNA expression and protein enzymatic activity were not detectable in preadipocytes. However, upon differentiation, renin mRNA and both intracellular and extracellular renin activity were upregulated. In differentiated adipocytes, forskolin treatment resulted in a 28-fold increase in renin mRNA, whereas TNF[alpha] treatment decreased renin mRNA fourfold. IL-6, insulin, and angiotensin (Ang) II were without effect. In contrast, forskolin and TNF[alpha] each increased renin protein secretion 12-and sevenfold, respectively. Although both forskolin and TNF[alpha] induce lipolysis in adipocytes, fatty acids, prostaglandin [E.sub.2], and lipopolysaccharide had no effect on renin mRNA or secretion. To evaluate the mechanism(s) by which forskolin and/or TNF[alpha] are able to regulate renin secretion, a general lipase inhibitor (E600) and PKA inhibitor (H89) were used. Both inhibitors attenuated forskolin-induced renin release, whereas they had no effect on TNF[alpha]-regulated secretion. In contrast, E600 potentiated forskolin-stimulated renin mRNA levels, whereas H89 had no effect. Neither inhibitor had any influence on TNF[alpha] regulation of renin mRNA. Relative to lean controls, renin expression was reduced 78% in the epididymal adipose tissue of obese male C57B1/6J mice, consistent with TNF[alpha]-mediated downregulation of renin mRNA in the culture system. In conclusion, the expression and secretion of renin are regulated under a complex series of hormonal and metabolic determinants in mature 3T3-L1 adipocytes. secretion; tumor necrosis factor-[alpha]; forskolin; renin-angiotensin system; angiotensin II

Oxidative stress plays an important role in obesity-related metabolic diseases. Glutathione peroxidase (GPX) is an antioxidant enzyme downregulated in adipose tissue of obese mice. However, the role of GPX in adipocytes remains elusive. The objective of this study was to clarify the pathophysiological changes in GPX activity and glutathione metabolism and their roles in the pathogenesis of insulin resistance in adipocytes. To achieve this goal, we measured cellular GPX activity, glutathione (GSH) contents, GSH/GSSG ratio, and mRNA expression of [gamma]-glutamylcysteine synthetase ([gamma]-GCS), a rate-limiting enzyme for de novo GSH synthesis, in adipose tissue of control and ob/ob mice and in 3T3-L1 adipocytes treated with insulin, [H.sub.2][O.sub.2], free fatty acid (FFA), or TNF[alpha]. Furthermore, we investigated the effects of GPX inhibition with a specific GPX inhibitor or RNA interference against GPX, [H.sub.2][O.sub.2], and reduced GSH on insulin signaling in 3T3-L1 adipocytes, ob/ob Mice showed not only a decrease in cellular activity of GPXs (GPX1, -4, and -7) but also an increase in [gamma]-GCS expression, resulting in increased GSH contents in adipose tissue. These alterations in glutathione metabolism were also observed during differentiation of 3T3-L1 cells and their exposure to insulin, FFA ,or [H.sub.2][O.sub.2]. Inhibition of GPX activity, addition of GSH, and [H.sub.2][O.sub.2] resulted in impaired insulin signaling in 3T3-L1 adipocytes. These results suggest that decreased GPX activity and increased [gamma]-GCS expression lead to overaccumulation of GSH, which might be involved in the pathogenesis of insulin resistance in obesity. glutathione peroxidase; [gamma]-glutamylcysteine synthetase; oxidative stress; insulin resistance

Understanding preadipocyte differentiation in economically important adipose depots will facilitate efforts to selectively increase intramuscular (i.m.) lipid accretion in cattle. The objectives of this study were to determine if glucocorticoid receptor (GR) expression differs among bovine stromal-vascular (S-V) cells derived from i.m., subcutaneous (s.c.), and perirenal (p.r.) adipose tissue, and to evaluate the effects of dexamethasone (DEX) on adipogenesis of these cell populations. Stromal-vascular cells isolated from i.m., s.c., and p.r. adipose tissues of 2 steers were propagated in culture and exposed to 0 or 250 nM DEX for 48 h. Cell lysates were subjected to GR immunoblot analysis, and immunoreactive protein bands of 97, 62, and 48 kDa were detected and expressed relative to [beta]-actin immunoreactivity. The abundance of each GR immunoreactive protein was similar among S-V cell populations (P > 0.50). Dexamethasone exposure decreased the abundance of the 97 and 62 kDa GR immunoreactive bands in S-V cells from the 3 depots (P < 0.001), but did not affect the expression of the 48 kDa band (P = 0.96). Stromal-vascular cells isolated from 3 steers were grown in culture, and upon confluence, were exposed to 0. 25, or 2,500 nM DEX for 48 h. After an additional 10 d in differentiation media, differentiation was determined by glycerol-3-phosphate dehydrogenase (GPDH) specific activity and oil red O staining. The extent of differentiation differed by depot (p.r. > s.c. > i.m.; P < 0.05). Compared with control, 2,500 nMDEX increased GPDH activity in S-V cells from all depots (P < 0.05), and no interaction between depot and DEX concentration was observed (P = 0.99). We observed an adipose tissue depot by DEX concentration interaction (P = 0.03) for S-V cells with large ([greater than or equal to] 10 [micro]m-diameter) lipid droplets. The percentage of p.r. S-V cells with large lipid droplets increased in response to DEX in a linear manner (P < 0.02), but only increased greater than control in s.c. cells exposed to 2,500 nM DEX (P = 0.002). Dexamethasone did not significantly increase the percentage of i.m. S-V cells with large lipid droplets (P > 0.27). Collectively. these data demonstrate differences in adipogenic activity among bovine i.m., s.c., and p.r. S-V cells, but indicate no relationship between adipogenic activity and glucocorticoid receptor abundance or function. Key words: adipogenesis, bovine, dexamethasone, differentiation, glucocorticoid receptor, stromal-vascular cell

Leptin has profound effects on adipose tissue metabolism. However, it remains unclear whether direct leptin signaling in adipocytes is involved. We addressed this question by transplanting inguinal adipose tissue stromal vascular cells (SVCs) from 4- to 5-wk-old wild-type (WT) and leptin receptor-deficient [[Lepr.sup.db/db] (db)] mice to inguinal and sternal subcutaneous sites in Ncr nude mice. Both WT and db SVCs gave rise to mature adipocytes with normal morphologies 3 mo after the transplantation. The average adipocyte size ([micro][m.sup.2]/cell) was not significantly different between WT and db transplants at either the inguinal (1,630 [+ or -] 103 vs. 1,491 [+ or -] 74) or the sternal site (1,788 [+ or -] 107 vs. 1,596 [+ or -] 92). Expression levels of [[beta].sub.3]-adrenergic receptor, a major mediator of lipid mobilization, were indistinguishable between WT and db transplants and similar to those of the hosts. Additionally, adipocyte sizes of inguinal transplants and endogenous inguinal adipose tissues were closely correlated ([beta] = 0.76, P < 0.001), suggesting that the metabolic milieu of host mice has significant effects on adipocyte size of the transplants. Contrary to the indifference to donor's Lepr genotype, adipocyte size of the transplants was significantly affected by the donor's sex in a leptin receptor-dependent manner. In WT transplants, female SVCs gave rise to smaller adipocytes than male SVCs (1,358 [+ or -] 127 vs. 2,133 [+ or -] 171, P < 0.05). However, this sex difference was not significant in db transplants (1,537 [+ or -] 121 vs. 1,655 [+ or -] 140, P = 0.22). These data suggest that: 1) long-form receptor-mediated direct leptin signaling has no significant cell-autonomous effect on adipocyte differentiation and metabolism in adult mice, 2) sex may affect adipocyte metabolism via genetic and/or epigenetic programming, and 3) leptin may potentiate sexual dimorphism in adipocyte metabolism. [[beta].sub.3]-adrenergic receptor; sex; genetic and epigenetic programming; adipocyte progenitor cells; db mutation

White adipose tissue is intimately involved in the regulation of immunity and inflammation. We reported that human mesentefic preadipocytes express the substance P (SP)-mediated neurokinin-1 receptor (NK-1R), which signals proinflammatory responses. Here we tested the hypothesis that SP promotes proliferation and survival of human mesenteric preadipocytes and investigated responsible mechanism(s). Preadipocytes were isolated from mesenteric fat biopsies during gastric bypass surgery. Proliferative and antiapoptotic responses were delineated in 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetra-zolium (MTS), bromodeoxyuridine (BrdU), caspase-3, and TUNEL assays, as well as Western immunoanalysis. SP ([10.sup.-7] M) increased MTS and proliferation (BrdU) and time dependently (15-30 min) induced Akt, EGF receptor, IGF receptor, integrin [alpha]V[beta]3, phosphatidylinositol 3-kinase, and PKC-[theta] phosphorylation. Furthermore, pharmacological antagonism of Akt and PKC-[theta] activation significantly attenuated SP-induced preadipocyte proliferation. Exposure of preadipocytes to the proapoptotic Fas ligand (FasL, 100 [micro]M) resulted in nuclear DNA fragmentation (TUNEL assay), as well as increased cleaved poly (ADP-ribose) polymerase, cleaved caspase-7, and caspase-3 expression. Cotreatment with SP almost completely abolished these responses in a NK-1R-dependent fashion. SP ([10.sup.-7] M) also time dependently stimulated expression 4E binding protein 1 and phosphorylation of p70 S6 kinase, which increased protein translation efficiency. SP increases preadipocyte viability, reduces apoptosis, and stimulates proliferation, possibly via cell cycle upregulation and increased protein translation efficiency. SP-induced proliferative and antiapoptotic pathways in fat depots may contribute to development of the creeping fat and inflammation characteristic of Crohn's disease. neuropeptide; creeping fat; Crohn's disease; adipocyte; proliferation; protein kinase C-[theta]; Akt

Adipocytes isolated from apolipoprotein E (apoE)-knockout (EKO) mice display alterations in triglyceride (TG) metabolism and gene expression. The present studies were undertaken to evaluate the impact of endogenously produced adipocyte apoE on these adipocyte parameters in vivo, independent of the profoundly disturbed metabolic milieu of EKO mice. Adipose tissue from wild-type (WT) or EKO mice was transplanted into WT recipients, which were then fed chow or high-fat diet for 8-10 wk. After a chow diet, freshly isolated transplanted EKO adipocytes were significantly (P < 0.05) smaller (70%) than transplanted WT adipocytes and displayed significantly lower rates of TG synthesis and higher rates of TG hydrolysis. Transplanted EKO adipocytes also had higher mRNA levels for adiponectin, perilipin, and genes coding for enzymes in the fatty acid oxidation pathway and lower levels of caveolin. After a high-fat diet and consequent increase in circulating lipid and apoE levels, transplanted WT adipocyte size increased by 106 x [10.sup.3] [micro][m.sup.3], whereas EKO adipocyte size increased only by 19 x [10.sup.3] [micro][m.sup.3]. Endogenous host adipose tissue harvested from WT recipients of transplanted WT or EKO adipose tissue did not demonstrate any difference in adipocyte size. Consistent with the in vivo observations, EKO adipocytes synthesized less TG when incubated with apoE-containing TG-rich lipoproteins than WT adipocytes. Our results establish a novel in vivo role for endogenously produced apoE, distinct from circulating apoE, in modulation of adipocyte TG metabolism and gene expression. They support a model in which endogenously produced adipocyte apoE facilitates adipocyte lipid acquisition from circulating TG-rich lipoproteins. obesity; adipose tissue; apolipoproteins

Infection and inflammation affect adipose triglyceride metabolism, resulting in increased plasma free fatty acid (FFA) and VLDL levels during the acute-phase response. Lipin-1, a multifunctional protein, plays a critical role in adipose differentiation, mitochondrial oxidation, and triglyceride synthesis. Here, we examined whether LPS [a Toll-like receptor (TLR)-4 activator], zymosan (a TLR-2 activator), and proinflammatory cytokines regulate lipin-1 in adipose tissue. LPS administration caused a marked decrease in the levels of lipin-1 mRNA and protein in adipose tissue. The decrease in lipin-1 mRNA levels occurred rapidly and lasted for at least 24 h. In contrast, lipin-2 and -3 mRNA levels did not change, suggesting specific repression of lipin-1. Zymosan similarly decreased lipin-1 mRNA without affecting lipin-2 or lipin-3 mRNA levels. To determine the pathways by which LPS repressed lipin-1, we examined the effect of proinflammatory cytokines on cultured adipocytes. In 3T3-L1 adipocytes, TNF-[alpha], IL-1[beta], and IFN-[gamma], but not LPS or IL-6, caused a decrease in lipin-1 mRNA levels. Furthermore, TNF-[alpha] and IL-1[beta] administration also decreased mRNA levels of lipin-1 in adipose tissue in mice. Importantly, the LPS-induced decrease in lipin-1 mRNA levels was significantly but not totally blunted in TNF-[alpha]/IL-1 receptor-null mice compared with controls, suggesting key roles for TNF-[alpha]/IL-1[beta] and other cytokines in mediating LPS-induced repression of lipin-1. Together, our results demonstrate that expression of lipin-1, one of the essential triglyceride synthetic enzymes, was suppressed by LPS, zymosan, and proinflammatory cytokines in mouse adipose tissue and in cultured 3T3-L1 adipocytes, which could contribute to a decrease in the utilization of FFA to synthesize triglycerides in adipose tissue, thus promoting the release of FFA into the circulation. inflammation; peroxisome proliferator-activated receptor; zymosan; tumor necrosis factor; interleukin-1; fatty acids; lipopolysaccharide

NCX 4016 is a nitric oxide (NO)-donating derivative of acetylsalicylic acid. NO and salicylate, in vivo metabolites of NCX 4016, were shown to be potential actors in controlling glucose homeostasis. In this study, we evaluated the action of NCX 4016 on the capacity of 3T3-L1 adipocytes to transport glucose in basal and insulin-stimulated conditions. NCX 4016 induced a twofold increase in glucose uptake in parallel with the translocation of the glucose transporters GLUT1 and GLUT4 to the plasma membrane, leaving unaffected their total expression levels. Importantly, NCX 4016 further increased glucose transport induced by a physiological concentration of insulin. The stimulatory effect of NCX 4016 on glucose uptake appears to be mediated by its NO moiety. Indeed, it is inhibited by a NO scavenger and treatment with acetylsalicylic or salicylic acid had no effect. Although NO is involved in the action of NCX 4016, it did not mainly depend on the soluble cGMP cyclase/ protein kinase G pathway. Furthermore, NCX 4016-stimulated glucose transport did not involve the insulin-signaling cascade required to stimulate glucose transport. NCX 4016 induces a small activation of the mitogen-activated protein kinases p38 and c-Jun N[H.sub.2]-terminal kinase and no activation of other stress-activated signaling molecules, including extracellular signal-regulated kinase, inhibitory factor [kappa]B, or AMP-activated kinases. Interestingly, NCX 4016 modified the content of S-nitrosylated proteins in adipocytes. Taken together, our results indicate that NCX 4016 induced glucose transport in adipocytes through a novel mechanism possibly involving S-nitrosylation. NCX 4016 thus possesses interesting characteristics to be considered as a candidate molecule for the treatment of patients suffering from metabolic syndrome and type 2 diabetes. nitric oxide donation; adipocytes; glucose transporter translocation; S-nitrosylation; diabetes

Obesity is increasing in an epidemic manner in most countries and constitutes a public health problem by enhancing the risk for cardiovascular disease and metabolic disorders such as type 2 [...]

A dual-tracer approach (dietary [sup.14]C-palmitate and intraperitoneal [sup.3]H-[H.sub.2]O) was used to assess the trafficking of dietary fat and net retention of carbon in triglyceride depots during the first 24 h of weight regain. Obesity-prone male Wistar rats were allowed to mature under obesogenic conditions for 16 wk. One group was switched to ad libitum feeding of a low-fat diet for 10 wk (Obese group). The remaining rats were switched to an energy-restricted, low-fat diet for 10 wk that reduced body weight by 14% and were then assessed in energy balance (Reduced group), with free access to the low-fat diet (Relapse-Dayl group), or with a provision that induced a minor imbalance (+ 10 kcal) equivalent to that observed in obese rats (Gap-Matched group). Fat oxidation remained at a high, steady rate throughout the day in Obese rats, but was suppressed in Reduced, Gap-Matched, and Relapse-Dayl rats though 9, 18, and 24 h, respectively. The same caloric excess in Obese and Gap-Matched rats led to less fat oxidation over the day and greater trafficking of dietary fat to visceral depots in the latter. In addition to trafficking nutrients to storage, Relapse-Dayl rats had more small, presumably new, adipocytes at the end of 24 h. Dietary fat oxidation at 24 h was related to the phospborylation of skeletal muscle acetyl-CoA carboxylase and fatty acid availability. These observations provide evidence of adaptations in the oxidation and trafficking of dietary fat that extend beyond the energy imbalance, which facilitate rapid, efficient regain during the relapse to obesity. acetyl-CoA carboxylase; metabolic inflexibility; postobese; adipocyte cellularity

Apelin, a novel peptide with significant cardioactive properties, is upregulated by insulin in adipocytes. However, the mechanism by which insulin promotes apelin production is unknown. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor involved in the angiogenic and metabolic responses to tissue hypoxia, has been shown to be activated by insulin in various settings. We therefore hypothesized that HIF-1 regulates insulin-mediated apelin expression in adipocytes. 3T3-L1 cells were differentiated into adipocytes in culture. For experiments, serum-starved 3T3-L1 cells were exposed to insulin and/or a 1% [O.sub.2] environment. Apelin expression was assessed using quantitative real-time PCR and ELISA. To directly assess the role of HIF-1 in apelin production, we differentiated mouse embryonic fibroblasts (MEFs) containing a targeted deletion of the HIF-1[alpha] gene into adipocytes and measured their response to insulin and hypoxia. Apelin expression in mature 3T3-L1 adipocytes was increased significantly by insulin and was attenuated by pharmacological inhibition of insulin signaling. Exposure of cells to either hypoxia or the chemical HIF activators cobalt chloride (CO[Cl.sub.2]) and dimethyloxaloylglycine (DMOG) resulted in significant upregulation of apelin, consistent with a role for HIF in apelin induction. Moreover, hypoxia-, CO[Cl.sub.2]-, DMOG-, and insulin-induced apelin expression were all attenuated in differentiated HIF-let-deficient MEFs. In summary, in cultured 3T3-L1 adipocytes and differentiated MEFs, HIF-1 appears to be involved in hypoxia-and insulin-induced apelin expression. hypoxia-inducible factor; adipocyte; obesity

The Wnt family of secreted glycoproteins had previously been shown to regulate diverse processes during early development. Wnt signaling also plays a key role in the homeostasis of adult tissues maintaining stem cell pluripotency and determining differentiating cell fate. The age-related decrease in Wnt signaling may contribute to increased muscle adiposity and diminished bone strength. In the current study, we investigated the long-term metabolic consequences of the upregulated Wnt/[beta]-catenin signaling in skeletal muscles of adult diet-induced obese (DIO) rats. To this end, we generated a recombinant adeno-associated virus (rAAV) vector encoding murine Wnt10b cDNA. The long-term expression of rAAV1-Wnt10b was tested after intramuscular injection in the female DIO rat. Animals fed high-fat diet and treated with rAAV1-Wnt10b showed a sustained reduction in body weight compared with controls, and expression of Wnt10b was accompanied by a reduction in hyperinsulinemia and triglyceride plasma levels as well as improved glucose homeostasis. Nuclear magnetic resonance methods revealed that ectopic expression of Wnt10b resulted in a decrease in both global and muscular fat deposits in DIO rats. The long-range effect of locally expressed Wnt10b was also manifested through the increased bone mineral density. The detailed analysis of molecular markers revealed fibroblast growth factor-4 and vascular endothelial growth factor as possible mediators of the systemic effect of Wnt10b transgene expression. Our data demonstrate that altering Wnt/[beta]-catenin signaling in the skeletal muscle of an adult animal invokes moderate responses with favorable metabolic profile, bringing the notion of alternative therapeutic modality in the treatment of obesity, diabetes, and osteoporosis. Wnt10b; gene therapy; obesity: diabetes

Central-omental obesity plays a causative role in the pathogenesis of the metabolic syndrome. Adipokines are involved in the pathogenesis of this syndrome. However, adipokines secreted by omental adipose tissue (OAT) are still poorly characterized in human obesity. Therefore, we searched for novel adipokines abnormally secreted by OAT in obesity and examined their relationships with some features of metabolic syndrome and the respective contribution of adipocytes vs. stromal-vascular cells. OAT from obese and nonobese men was fractionated into adipocytes and SV cells, which were then cultured. Medium was screened by medium-scale protein arrays and ELISAs. Adipokine mRNA levels were measured by real-time RT-qPCR. We detected 16 cytokines secreted by each cellular fraction of lean and obese subjects. Of the 16 cytokines, six adipokines were newly identified as secretory products of OAT, which were dysregulated in obesity: three chemokines (growth-related oncogen factor, RANTES, macrophage inflammatory protein-l[beta]), one interleukin (IL-7), one tissue inhibitor of metalloproteinases (TIMP-1), and one growth factor (thrombopoietin). Their secretion and expression were enhanced in obesity, with a relatively similar contribution of the two fractions. The higher proportion of macrophages and endothelial cells in obesity may contribute to this enhanced production as well as changes in intrinsic properties of hypertrophied adipocytes. Accordingly, mRNA concentrations of most of these adipokines increased during adipocyte differentiation. Eventually, expression of the investigated adipokines did correlate with several features of the metabolic syndrome. In conclusion, six adipokines were newly identified as oversecreted by OAT in obesity. These adipokines may link obesity to its cardiovascular or metabolic comorbidities. obesity; adipocytes; stromal-vascular cells; inflammation; metabolic syndrome

Triacylglycerol (TAG) stored in adipose tissue can be rapidly mobilized by the hydrolytic action of lipases, with the release of fatty acids (FA) that are used by other tissues during times of energy deprivation. Unlike synthesis of TAG, which occurs not only in adipose tissue but also in other tissues such as liver for very-low-density lipoprotein formation, hydrolysis of TAG, lipolysis, predominantly occurs in adipose tissue. Until recently, hormone-sensitive lipase was considered to be the key rate-limiting enzyme responsible for regulating TAG mobilization. However, recent studies on hormone-sensitive lipase-null mice have challenged such a concept. A novel lipase named desnutrin/ATGL has been recently discovered to play a key role in lipolysis in adipocytes. Lipolysis is under tight hormonal regulation. Although opposing regulation of lipolysis in adipose tissue by insulin and catecholamines is well understood, autocrine/paracrine factors may also participate in its regulation. Intricate cooperation of these endocrine and autocrine/ paracrine factors leads to a fine regulation of lipolysis in adipocytes, needed for energy homeostasis. In this review, we summarize and discuss the recent progress made in the regulation of adipocyte lipolysis. doi:10.1152/ajpgi.00554.2006 fatty acids; desnutrin/ATGL; hormone-sensitive lipase; catecholamines; insulin

Insulin resistance (IR) is typically more severe in obese individuals with type 2 diabetes (T2DM) than in similarly obese non-diabetics but whether there are group differences in body composition and whether such differences contribute to the more severe IR of T2DM is uncertain. DEXA and regional CT imaging were conducted to assess adipose tissue (AT) distribution and fat content in liver and muscle in 67 participants with T2DM (F39/M28, age 60 [+ or -] 7 yr, BMI 34 [+ or -] 3 kg/[m.sup.2]) and in 35 similarly obese, non-DM volunteers (F20/M15, age 55 [+ or -] 8 yr, BMI 33 [+ or -] 2 kg/[m.sup.2]). A biopsy of subcutaneous abdominal AT was done to measure adipocyte size. A glucose clamp was performed at an insulin infusion of 80 mU x [min.sup.-1] x [m.sup.-2]. There was more severe IR in T2DM (6.1 [+ or -] 2.3 vs. 9.9 [+ or -] 3.3 mg x [min.sup.1] x kg [FFM.sup.-1]; p < 0.01). Group comparisons of body composition parameters was performed after adjusting for the effect of age, gender, race, height and total fat mass (FM). T2DM was associated with less leg FM (-1.2 [+ or -] 0.4 kg, P < 0.01), more trunk FM (+1.1 [+ or -] 0.4 kg, P < 0.05), greater hepatic fat (P < 0.05), and more subfascial adipose tissue around skeletal muscle (P < 0.05). There was a significant group x sex interaction for VAT (P < 0.01), with greater VAT in women with T2DM (P < 0.01). Mean adipocyte size (AS) did not significantly differ across groups, and smaller AS was associated with increased leg FM, whereas larger AS was related to more trunk FM (both P < 0.05). Group differences in IR were less after adjusting for group differences in leg FM, trunk FM, and hepatic fat, but these adjustments only partially accounted for the greater severity of IR in T2DM. In summary, T2DM, compared with similarly obese nondiabetic men and women, is associated with less leg FM and greater trunk FM and hepatic fat. doi:10.1152/ajpendo.003934.2006 adipocytes; obesity; body composition; computed tomography

Adiponectin, a physiologically active polypeptide secreted by adipocytes, shows insulin-sensitizing, anti-inflammatory, and antiatherogenic properties in rodents and humans. To assess the effects of chronic hyperadiponectinemia on metabolic phenotypes, we established three lines of transgenic mice expressing human adiponectin in the liver. When maintained on a high-fat/high-sucrose diet, mice of two lines that had persistent hyperadiponectinemia exhibited significantly decreased weight gain associated with less fat accumulation and smaller adipocytes in both visceral and subcutaneous adipose tissues. Macrophage infiltration in adipose tissue was markedly suppressed in the transgenic mice. Expression levels of adiponectin receptors were not altered in skeletal muscle or liver. Circulating levels of endogenous adiponectin were elevated, whereas fasting glucose, insulin, and leptin levels were reduced compared with control mice. In the hyperadiponectinemic mice daily food intake was not altered, but oxygen consumption was significantly greater, suggesting increased energy expenditure. Moreover, high-calorie diet-induced premature death was almost completely prevented in the hyperadiponectinemic mice in association with attenuated oxidative DNA damage. The transgenic mice also showed longer life span on a conventional low-fat chow. In conclusion, transgenic expression of human adiponectin blocked the excessive fat accumulation and reduced the morbidity and mortality in mice fed a high-calorie diet. These observations may provide new insights into the prevention and therapy of metabolic syndrome in humans. adipocyte; macrophage; life span; 8-hyroxy-2-deoxyguanosine doi:10.1152/ajpendo.00645.2006

Apolipoprotein E (apoE) is a multifunctional protein that is highly expressed in human and murine adipose tissue. Endogenous adipocyte apoE expression influences adipocyte triglyceride turnover and modulates the expression of genes involved in lipid synthesis and oxidation. We now demonstrate the regulation of adipose tissue apoE expression by nutritional status in lean and obese mice. Obesity induced by high-fat diet, or by hyperphagia in ob/ob mice, produces significant reduction of adipose tissue apoE expression at the protein and messenger RNA level. Fasting in C57BL/6J mice for 24 h significantly increased apoE protein and messenger RNA levels. In ob/ob mice, transplantation of adipose tissue from lean littermate controls to restore circulating leptin levels produced significant weight loss over 12 wk and also produced an increase in adipose tissue apoE expression. The increase in adipose tissue apoE expression in this model, however, did not require leptin. Adipose tissue apoE was also significantly increased in ob/ob mice after a 48-h fast or after 7 days of caloric restriction. In summary, obesity suppresses adipose tissue apoE expression, whereas fasting or weight loss increases it. From our previous observations, these changes in adipose tissue apoE expression will have significant impact on adipose tissue lipid flux and lipoprotein metabolism. Furthermore, these results suggest adipose tissue apoE participates in defending adipose tissue and organismal energy homeostasis in response to nutritional perturbation. adipocytes; obesity doi:10.1152/ajpendo.00118.2007

Peroxisome proliferator-activated receptor-[gamma] (PPAR-[gamma]) plays a critical role in regulating insulin sensitivity and glucose homeostasis. In this study, we identified highly efficient small interfering RNA (siRNA) sequences and used lentiviral short hairpin RNA and electroporation of siRNAs to deplete PPAR-[gamma] from 3T3-LI adipocytes to elucidate its role in adipogenesis and insulin signaling. We show that PPAR-[gamma] knockdown prevented adipocyte differentiation but was not required for maintenance of the adipocyte differentiation state after the cells had undergone adipogenesis. We further demonstrate that PPAR-[gamma] suppression reduced insulin-stimulated glucose uptake without affecting the early insulin signaling steps in the adipocytes. Using dual siRNA strategies, we show that this effect of PPAR-[gamma] deletion was mediated by both GLUT4 and GLUT1. Interestingly, PPAR-[gamma]-depleted cells displayed enhanced inflammatory responses to TNF-[alpha] stimulation, consistent with a chronic anti-inflammatory effect of endogenous PPAR-[gamma]. In summary, 1) PPAR-[gamma] is essential for the process of adipocyte differentiation but is less necessary for maintenance of the differentiated state, 2) PPAR-[gamma] supports normal insulin-stimulated glucose transport, and 3) endogenous PPAR-[gamma] may play a role in suppression of the inflammatory pathway in 3T3-L1 cells. small interfering RNA; lentivirus; peroxisome proliferator activated-receptor-[gamma] doi:10.1152/ajpendo.00695.2006

Reactive oxygen species (ROS) production has recently been established as an essential contributor in the pathogenesis of obesity-associated insulin resistance. The FoxO1 pathway plays a role not only in nutrient sensing but also in regulating ROS production. We exposed adipocytes in free fatty acids (FFA) and demonstrated that FoxO1 protein levels decrease in a dose-dependent manner. The FoxO1 downregulation correlated with an increase in the production of ROS and a proinflammatory adipokine pattern characterized by a decrease in adiponectin and an increase in IL-6, plasminogen activator inhibitor-l, and monocyte chemotactic protein-1 mRNA expression levels. Similarly, a decrease in FoxO1 protein levels was seen in adipocytes of db/db mice compared with controls. Treatment with the sirtuin agonist resveratrol, which translocates FoxOl to the nucleus, increased FoxO1 protein levels in adipocytes exposed to FFA. This correlated with a decrease in the generation of ROS and a partial reversal of the proinflammatory adipokine pattern. Together these results indicate that the insulin-resistant adipocyte produced by the exposure to a high concentration of fatty acids is characterized by decreased levels of FoxOl. These data also suggest that modulation of the Sirtl/FoxO1 pathway is a potentially useful therapeutic target for the obesity-induced dysfunctional adipocyte. inflammatory cytokines; insulin resistance; fatty acids doi:10.1152/ajpendo.00629.2006.

Abstract Evidence of the signs associated with skin aging often first appears in the periorbital area and includes wrinkles, eyelid bags, circles around the eye, or a 'tired' look. This [...]

Previously, a small molecule, reversine, was identified that reverses lineage-committed murine myoblasts to a more primitive multipotent state. Here, we show that reversine can increase the plasticity of C2C12 myoblasts at the single-cell level and that reversine-treated cells gain the ability to differentiate into osteoblasts and adipocytes under lineage-specific inducing conditions. Moreover, reversine is active in multiple cell types, including 3T3E1 osteoblasts and human primary skeletal myoblasts. Biochemical and cellular experiments suggest that reversine functions as a dual inhibitor of nonmuscle myosin II heavy chain and MEK1, and that both activities are required for reversine's effect. Inhibition of MEK1 and nonmuscle myosin II heavy chain results in altered cell cycle and changes in histone acetylation status, but other factors also may contribute to the activity of reversine, including activation of the PI3K signaling pathway. myoblasts | multipotent | small molecule | osteoblast | adipocyte

Lipoprotein lipase (LPL) activity is necessary for adipocytes to take up triglycerides from the circulation, and regional differences in LPL activity could help determine regional fat storage. LPL activity has been reported to increase as a function of fat cell size, but this issue has not been extensively evaluated in different depots comparing sexes. Our objective was to determine whether sex alters the relationship between LPL activity and fat cell size. Subcutaneous adipose tissue biopsies were taken from the abdomen and thigh after an overnight fast and 1 h after a meal in 65 females (BMI 25.4 [+ or -] 0.8, means [+ or -] SE) and 41 males (BMI 23.7 [+ or -] 0.3); gluteal adipose samples were obtained in 47 of the females and 27 of the males. Fat cell size was greater in females than males in thigh (P < 0.005) and gluteal (P < 0.05) regions but not in the abdomen. There was a relationship between fasting LPL activity/ fat cell and fat cell size in females (abdomen [r.sup.2] = 0.52, P < 0.0001; gluteal [r.sup.2] = 0.23, P < 0.005; thigh [r.sup.2] = 0.19, P < 0.005). In males, this relationship was seen only in the abdomen ([r.sup.2] = 0.51, P < 0.0001) and thigh ([r.sup.2] = 0.17, P < 0.05). Males and females had a significantly different relationship in the thigh only in the fasted state. Similar results were found in the fed state, although the strength of the relationship decreased in the abdominal regions for females only. This suggests fundamental differences in the regulation of triglyceride uptake between males and females and adipose regions. lipoprotein lipase; body fat distribution; postprandial; adipose tissue biopsies doi: 10.1152/ajpendo.00601.2006

Fibroblast growth factor 21 (FGF21) is a liver-derived endocrine factor that stimulates glucose uptake in adipocytes. Here, we show that FGF21 activity depends on [beta]Klotho, a single-pass transmembrane protein whose expression is induced during differentiation from preadipocytes to adipocytes. [beta]Klotho physically interacts with FGF receptors 1c and 4, thereby increasing the ability of these FGF receptors to bind FGF21 and activate the MAP kinase cascade. Knockdown of [beta]Klotho expression by siRNA in adipocytes diminishes glucose uptake induced by FGF21. Importantly, administration of FGF21 into mice induces MAP kinase phosphorylation in white adipose tissue and not in tissues without [beta]Klotho expression. Thus, [beta]Klotho functions as a cofactor essential for FGF21 activity. Klotho | glucose | adipocyte | siRNA | GLUT1

Circulating adiponectin levels are increased by the thiazolidinedione (TZD) class of PPAR[gamma] agonists in concert with their insulin-sensitizing effects. Two receptors for adiponectin (AdipoR1 and AdipoR2) are widely expressed in many tissues, but their physiological significance to human insulin resistance remains to be fully elucidated. We examined the expression patterns of AdipoR1 and AdipoR2 in fat and skeletal muscle of human subjects, their relationship to insulin action, and whether they are regulated by TZDs. Expression patterns of both AdipoRs were similar in subcutaneous and omental fat depots, with higher expression in adipocytes than in stromal cells and macrophages. To determine the effects of TZDs on AdipoR expression, subcutaneous fat and quadriceps muscle were biopsied in 14 insulin-resistant subjects with type 2 diabetes mellitus after 45 mg pioglitazone or placebo for 21 days. This duration of pioglitazone improved insulin's suppression of glucose production by 41% and enhanced stimulation of glucose uptake by 27% in concert with increased gene expression and plasma levels of adiponectin. Pioglitazone did not affect AdipoR expression in muscle, whole fat, or cellular adipose fractions, and receptor expression did not correlate with baseline or TZD-enhanced insulin action. In summary, both adiponectin receptors are expressed in cellular fractions of human fat, particularly adipocytes. TZD administration for sufficient duration to improve insulin action and increase adiponectin levels did not affect expression of AdipoR1 or AdipoR2. Although TZDs probably exert many of their effects via adiponectin, changes in these receptors do not appear to be necessary for their insulin-sensitizing effects. insulin resistance; diabetes mellitus; adipose tissue doi: 10.1152/ajpendo.00312.2006.

Ciglitizone, a class of thiazolidinediones, acts as a potent activator of the adipose differentiation program in established preadipose cell lines. Thiazolidinediones have also been investigated in diabetic patients and have been reported to act as peroxisome proliferator-activated receptor-[gamma] ligands. Intramuscular adipogenesis or marbling through transdifferentiation of satellite cells in cattle was successfully conducted earlier. In this report, the effects of ciglitizone on the differentiation pathway of porcine myogenic satellite cells was investigated. Semitendinosus muscle was aseptically taken from 10-d-old piglets under general anesthesia, and porcine satellite cells were obtained and grown to near confluence. Postconfluent cells (d 0) were further cultured in differentiation medium containing an adipogenic mixture plus ciglitizone (10 [micro]M) for 48 h. From d 2 onward, the cells were cultured only in the presence of ciglitizone until d 10. Controls were cultured in differentiation medium only. Exposure of porcine satellite cells to the adipogenic mixture plus ciglitizone generated lipid droplets on d 2, and subsequently, exposure of cells to ciglitizone alone helped in cytoplasmic lipid filling, providing them with the acquisition of adipocyte morphology. An increase (P < 0.05) in the fusion (structures containing 2 to 3 nuclei) of satellite cells was observed, and myosin heavy chain appeared with greater intensity (immunohistochemistry) in the control group from d 2 onward. Adipocytespecific transcriptional factors (i.e., CCAAT/enhancer binding protein-[alpha] and peroxisome proliferator-activated receptor-[gamma] were predominant during transdifferentiation and were observed with immuhistochemistry, Western blot (-47.2 and -60.4 kDa, respectively), and real-time PCR. Ciglitizone appeared to convert the differentiation pathway of satellite cells into that of adipoblasts. Key words: adipogenesis, ciglitizone, CCAAT/enhancer binding protein-[alpha], myogenesis, peroxisome proliferator-activated receptor-[gamma]

A clinically employed antihyperlipidemic drug, bezafibrate, has been characterized as a PPAR([alpha], -[gamma] and -[delta]) pan-agonist in vitro. Recent extended trials have highlighted its antidiabetic properties in humans. However, the underlying molecular mechanism is not fully elucidated. The present study was designed to explore potential regulatory mechanisms of intracellular glucocorticoid reactivating enzyme, 11[beta]-HSD1 and anti-diabetic hormone, adiponectin by bezafibrate in murine adipose tissue, and cultured adipocytes. Treatment of db/db mice with bezafibrate significantly ameliorated hyperglycemia and insulin resistance, accompanied by a marked reduction of triglyceride and nonesterified fatty acids. Despite equipotent in lipid-lowering effects, another fibrate, fenofibrate, did not show such beneficial effects on glycemic control. Treatment of bezafibrate caused a marked decrease in the mRNA level of 11[beta]-HSD1 preferentially in adipose tissue of db/db mice (-47%, P < 0.05), concomitant with a significant increase in plasma adiponectin level (+37%, P < 0.01). Notably, treatment of bezafibrate caused a marked decrease in the mRNA level (-34%, P < 0.01) and enzyme activity (-32%, P < 0.01) of 11[3-HSD1, whereas the treatment substantially augmented the expression (+71%, P < 0.01) and secretion (+27%, P < 0.01) of adiponectin in 3T3-L1 adipocytes. Knockdown of 11[beta]-HSD1 by siRNA confirmed that 11[beta]-HSD1 acts as a distinct oxoreductase in adipocytes and validated the enzyme activity assays in the present study. Effects of bezafibrate on regulation of 11[beta]-HSD1 and adiponectin in murine adipocytes were comparable with those in thiazolidinediones. This is the first demonstration that bezafibrate directly regulates 11[beta]-HSD 1 and adiponectin in murine adipocytes, both of which may contribute to metabolicallybeneficial effects by bezafibrate. metabolic syndrome; adiponectin

Adiponectin is an adipocyte-specific secretory hormone that can increase insulin sensitivity and promote adipocyte differentiation. Administration of adiponectin to obese or diabetic mice reduces plasma glucose and free fatty acid levels. Green tea polyphenols possess many pharmacological activities such as antioxidant, anti-inflammatory, antiobesity, and antidiabetic activities. To investigate whether green tea polyphenols have an effect on the regulation of adiponectin, we measured expression and secretion levels of adiponectin protein after treatment of each green tea polyphenols in 3T3-L1 adipocytes. We found that (-)-catechin enhanced the expression and secretion of adiponectin protein in a dose- and time-dependent manner. Furthermore, treatment of (-)-catechin increased insulin-dependent glucose uptake in differentiated adipocytes and augmented the expression of adipogenic marker genes, including PPART, CEBP[alpha], FAS, and SCD-1, when (-)-catechin was treated during adipocyte differentiation. In search of the molecular mechanism responsible for inducible effect of (-)-catechin on adiponectin expression, we found that (-)-catechin markedly suppresses the expression of Kruppel-like factor 7 (KLF7) protein, which has recently been reported to inhibit the expression of adiponectin and other adipogenesis related genes, including leptin, PPAR[gamma] C/EBP[alpha], and aP2 in adipocytes. KLF7 is a transcription factor in adipocyte and plays an important role in the pathogenesis of type 2 diabetes. Taken together, these data suggest that the upregulation of adiponectin protein by (-)-catechin may involve, at least in part, suppression of KLF7 in 3T3-L1 cells.

GLUT4 promoter activity is regulated by hormonal, metabolic, and tissue-specific controls. This complicates the study of GLUT4 gene transcription, as no cell culture model adequately recapitulates these extracellular regulators. While investigating cultured primary adipocytes as a model system for GLUT4 transcription, we observed that GLUT4 mRNA was specifically and rapidly downregulated upon tissue dispersal. Downregulation of GLUT4 mRNA was mediated in part by loss of regulatory control by the trans-acting factors that control GLUT4 transcriptional activity [the myocyte enhancer factor 2 (MEF2) transcription factor family and the GLUT4 enhancer factor] and their cognate DNA binding sites in transgenic mice. The differences in GLUT4 transcription when whole adipose tissue and cell culture model systems are compared can be correlated to a posttranslational phosphorylation of the transcription factor MEF2A. The difference in the MEF2A phosphorylation state in whole tissue vs. isolated cells may provide a further basis for the development of an in vitro system that could recapitulate fully regulated GLUT4 promoter activity. Development of an in vitro system to reconstitute GLUT4 transcriptional regulation will further efforts to discern the molecular mechanisms that underlie GLUT4 expression. glucose transporter-4 transcription; myocyte enhancer factor 2

Attainment of a brown adipocyte cell phenotype in white adipocytes, with their abundant mitochondria and increased energy expenditure potential, is a legitimate strategy for combating obesity. The unique transcriptional regulators of the primary brown adipocyte phenotype are unknown, limiting our ability to promote brown adipogenesis over white. In the present work, we used microarray analysis strategies to study primary preadipocytes, and we made the striking discovery that brown preadipocytes demonstrate a myogenic transcriptional signature, whereas both brown and white primary preadipocytes demonstrate signatures distinct from those found in immortalized adipogenic models. We found a plausible SIRT1-related transcriptional signature during brown adipocyte differentiation that may contribute to silencing the myogenic signature. In contrast to brown preadipocytes or skeletal muscle cells, white preadipocytes express Tcf21, a transcription factor that has been shown to suppress myogenesis and nuclear receptor activity. In addition, we identified a number of developmental genes that are differentially expressed between brown and white preadipocytes and that have recently been implicated in human obesity. The interlinkage between the myocyte and the brown preadipocyte confirms the distinct origin for brown versus white adipose tissue and also represents a plausible explanation as to why brown adipocytes ultimately specialize in lipid catabolism rather than storage, much like oxidative skeletal muscle tissue. microarray | myocyte | principal component analysis | differentiation | transcriptome

Adipocytes express the rate-limiting enzymes required for glycogen metabolism and increase glycogen synthesis in response to insulin. However, the physiological function of adipocytic glycogen in vivo is unclear, clue in part to the low absolute levels and the apparent biophysical constraints of adipocyte morphology on glycogen accumulation. To further study the regulation of glycogen metabolism in adipose tissue, transgenic mice were generated that overexpressed the protein phosphatase-1 (PP1) glycogen-targeting subunit (PTG) driven by the adipocyte fatty acid binding protein (aP2) promoter. Exogenous PTG was detected in gonadal, perirenal, and brown fat depots, but it was not detected in any other tissue examined. PTG overexpression resulted in a modest redistribution of PPl to glycogen particles, corresponding to a threefold increase in the glycogen synthase activity ratio. Glycogen synthase protein levels were also increased twofold, resulting in a combined greater than sixfold enhancement of basal glycogen synthase specific activity. Adipocytic glycogen levels were increased 200- to 400-fold in transgenic animals, and this increase was maintained to 1 yr of age. In contrast, lipid metabolism in transgenic adipose tissue was not significantly altered, as assessed by lipogenic rates, weight gain on normal or high-fat diets, or circulating free fatty acid levels after a fast. However, circulating and adipocytic leptin levels were doubled in transgenic animals, whereas adiponectin expression was unchanged. Cumulatively, these data indicate that murine adipocytes are capable of storing far higher levels of glycogen than previously reported. Furthermore, these results were obtained by overexpression of an endogenous adipocytic protein, suggesting that mechanisms may exist in vivo to maintain adipocytic glycogen storage at a physiological set point. insulin; glycogen synthesis: lipogenesis; protein phosphatase-1; targeting subunit

3T3-L 1 adipocytes develop insulin-resistant glucose transport upon preincubation with high (25 mM) glucose, provided that insulin (0.6 nM) is included, Akt activation is impaired, and high glucose and insulin act synergistically. Considerable evidence suggests that increased glucose flux via the hexosamine biosynthesis pathway enhances the O-GlcNAc modification (O-GlcNAcylation) of some critical protein(s) that may contribute to insulin resistance. However, whether enhanced protein O-GlcNAcylation is necessary for the development of insulin resistance is unknown. We used two strategies to test this hypothesis. The first strategy was the overexpression of O-GlcNAcase, which removes O-GlcNAc from Ser/Thr of proteins. Cells were infected with O-GlcNAcase-expressing adenovirus (or empty virus) 5 days before they were submitted to protocols that elicit (or not) insulin resistance. O-GlcNAcase was highly expressed and functional as assessed by Western blot, O-GlcNAcase assay, and marked reduction of O-GlcNAcylated proteins. The activity was mainly cytosolic. The second strategy was the expression of O-GlcNAc transferase (OGT) being markedly reduced by transfection of OGT siRNA, resulting in an approximately 90% decrease of nuclear and cytosolic OGT protein expression and similar reduction in O-GlcNAcylated proteins. Nontargeting siRNA had no effect. Preincubation in high glucose with low-dose insulin decreased the acute insulin response of glucose transport by at least 50% and impaired Akt activation. None of these parameters were affected by overexpression of O-GlcNAcase or by OGT knockout. Excess O-GlcNAcylation is one of many factors that can cause insulin resistance. It does not seem to be required for the development of glucose/insulin-induced insulin resistance of glucose transport and Akt activation in 3T3-L1 adipocytes. glucose transport; Akt activation; O-linked N-acetylglucosamine