3T3-F442A Cells Research Articles

No functional role of plasminogen activator inhibitor-1 in murine adipogenesis or adipocyte differentiation.

To substantiate a potential role of plasminogen activator inhibitor-1 (PAI-1) in adipogenesis, we have studied its effects on in vitro adipocyte differentiation and on in vivo adipose tissue formation. Our in vitro data do not support a functional role of PAI-1, as substantiated by our findings that: (i) inhibition of PAI-1 with a neutralizing antibody did not affect differentiation of 3T3-F442A preadipocytes; (ii) overexpression of murine PAI-1 in 3T3-F442A cells had no effect on differentiation; and (iii) differentiation of PAI-1-deficient murine embryonic fibroblasts into mature adipocytes was comparable to wild-type (WT) cells. Furthermore, our in vivo studies did not reveal an important role for PAI-1, as suggested by our findings that: (i) de novo fat pad formation in NUDE mice following injection of 3T3-F442A cells was not affected by a PAI-1 neutralizing antibody; and (ii) adipose tissue formation following combined injection of Matrigel and basic fibroblast growth factor was comparable in WT and in PAI-1 deficient mice. Taken together, these in vitro and in vivo studies in murine model systems do not support an important functional role of PAI-1 in adipogenesis.

Sulfonylurea Agents Exhibit Peroxisome Proliferator-activated Receptor γ Agonistic Activity

Sulfonylurea (SU) agents, including glimepiride and glibenclamide, are the most widely used oral hypoglycemic drugs, which stimulate insulin secretion primarily by binding to the SU receptor on the plasma membrane of pancreatic beta-cells. Thiazolidinediones, such as pioglitazone and rosiglitazone, are other hypoglycemic agents that effectively improve peripheral insulin resistance through activation of peroxisome proliferator-activated receptor gamma (PPARgamma). In the present study, we found that glimepiride specifically induced the transcriptional activity of PPARgamma in luciferase reporter assays. Glimepiride enhanced the recruitment of coactivator DRIP205 and dissociation of corepressors such as nuclear receptor corepressor and silencing mediator for retinoid and thyroid hormone receptors. In addition, glimepride directly bound to PPARgamma in a manner competitive to rosiglitazone, which is a proven ligand for PPARgamma. Furthermore, in 3T3-L1 adipocytes, glimepiride stimulated the transcriptional activity of the gene promoter containing PPAR-responsive element and altered mRNA levels of PPARgamma target genes including aP2, leptin, and adiponectin. Finally, glimepiride induced adipose differentiation in 3T3-F442A cells, which was known to differentiate into adipocytes in a PPARgamma-dependent manner. Most effects observed with glimepiride were also seen with glibenclamide. These data strongly suggest that glimepiride and glibenclamide, both of which belong to SU agents, should have PPARgamma agonist activity, whose potencies were 16-25% of the maximum level achieved by pioglitazone. Our observation that glimepiride and glibenclamide could act not only on SU receptor but also on PPARgamma may give an important clue to the development of novel antidiabetic drugs, which can enhance both insulin secretion from pancreatic beta-cells and peripheral insulin sensitivity.

Growth Hormone Receptor Is a Target for Presenilin-dependent γ-Secretase Cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.

Lysophosphatidic Acid Inhibits Adipocyte Differentiation via Lysophosphatidic Acid 1 Receptor-dependent Down-regulation of Peroxisome Proliferator-activated Receptor γ2

Lysophosphatidic acid (LPA) is a bioactive phospholipid acting via specific G protein-coupled receptors that is synthesized at the extracellular face of adipocytes by a secreted lysophospholipase D (autotaxin). Preadipocytes mainly express the LPA(1) receptor subtype, and LPA increases their proliferation. In monocytes and CV1 cells LPA was recently reported to bind and activate peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor also known to play a pivotal role in adipogenesis. Here we show that, unlike the PPARgamma agonist rosiglitazone, LPA was unable to increase transcription of PPARgamma-sensitive genes (PEPCK and ALBP) in the mouse preadipose cell line 3T3F442A. In contrast, treatment with LPA decreased PPARgamma2 expression, impaired the response of PPARgamma-sensitive genes to rosiglitazone, reduced triglyceride accumulation, and reduced the expression of adipocyte mRNA markers. The anti-adipogenic activity of LPA was also observed in the human SGBS (Simpson-Golabi-Behmel syndrome) preadipocyte cell line, as well as in primary preadipocytes isolated from wild type mice. Conversely, the anti-adipogenic activity of LPA was not observed in primary preadipocytes from LPA(1) receptor knock-out mice, which, in parallel, exhibited a higher adiposity than wild type mice. In conclusion, LPA does not behave as a potent PPARgamma agonist in adipocytes but, conversely, inhibits PPARgamma expression and adipogenesis via LPA(1) receptor activation. The local production of LPA may exert a tonic inhibitory effect on the development of adipose tissue.

The vasoactive peptide adrenomedullin is secreted by adipocytes and inhibits lipolysis through NO‐mediated β‐adrenergic agonist oxidation

Adipocytes are known to secrete a number of adipokines, but many adipocyte secretions and their functional importance remain to be characterized. This work shows that human white adipocytes and 3T3-F442A-derived adipocytes produce adrenomedullin (AM) and that AM acts in an autocrine/paracrine way on lipid metabolism by extracellular inactivation of isoproterenol, a beta-adrenergic agonist. AM is described as a counter-regulatory factor involved in the control of cardiovascular homeostasis. This peptide is believed to protect the heart from several complications implicated in obesity-linked cardiomorbidity, such as arterial hypertension, cardiac fibrosis, and decreased sinusal variability. The exact source of circulating AM remains a matter of debate, although endothelial and vascular smooth muscle cells seem to be important sites of production. We show that human adipose cells and 3T3-F442A-derived adipocytes express AM receptors and secrete AM. The function of this feature was investigated in 3T3-F442A cell line at the level of lipolysis regulation. AM inhibited beta-adrenergic-stimulated lipolysis by a nitric oxide (NO)-dependent mechanism, inducing a significant decrease in pD2 value for isoproterenol (8.6 +/- 0.2 vs. 9.8 +/- 0.1, P<0.001). This effect is cGMP-independent since it occurred in the presence of the NO-sensitive guanylate cyclase inhibitor ODQ. It is apparently mediated by a novel extracellular mechanism. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrated that AM-produced NO oxidized isoproterenol to generate its aminochrome, namely isoprenochrome. Isoprenochrome amounts were increased 3.62 +/- 1.13-fold in cell culture media (P<0.05). We describe for the first time that AM down-regulates lipolysis in adipocytes through the chemical modification of a beta-agonist.

Role of Gas-6 in Adipogenesis and Nutritionally Induced Adipose Tissue Development in Mice

A potential role of growth arrest-specific gene 6 (Gas-6) in energy storage in adipose tissue was investigated in murine models of obesity. Gas-6 is a ligand for the Axl, C-Mer, and Sky family of tyrosine kinase receptors. Whereas Gas-6, C-Mer, and Sky were expressed in mature murine adipocytes, the expression of Axl was restricted to the stromal-vascular fraction, which includes pre-adipocytes. During the in vitro conversion of adipogenic 3T3-F442A cells into mature adipocytes, the expression of Gas-6 increased in undifferentiated confluent pre-adipocytes during a transient phase of growth arrest. On treatment of these cells with an adipogenic medium, Gas-6 expression decreased sharply, coinciding with expression of early adipocytes markers. This modulation was not observed in the nonadipogenic 3T3-C2 cells. The Gas-6 mRNA level was transiently downregulated during nutritionally induced expansion of adipose tissues in vivo. When kept on a standard diet, no significant difference in either total body weight or weight of gonadal or subcutaneous fat pads was observed between Gas-6 deficient and wild-type mice. On exposure to a high-fat diet, however, Gas-6-deficient mice had significantly less fat mass than their wild-type counterparts. Gas-6 enhances the accumulation of adipose tissue in diet-induced obese mice.

THE JANUS* FACES OF ADRENOCEPTORS: FACTORS CONTROLLING THE COUPLING OF ADRENOCEPTORS TO MULTIPLE SIGNAL TRANSDUCTION PATHWAYS

1. The adrenoceptors (AR) are an important subfamily of rhodopsin-like G-protein-coupled receptors that couple to an increasingly large number of signalling mechanisms. Two important factors that determine the pathways that are used are the C-terminal region of the receptor and the agonist used to activate the receptor. 2. Studies of splice variants of the mouse beta3-AR showed that the C-terminus is a factor controlling the signalling characteristics. Although these receptors differ only at the C-terminus, the beta3b-AR coupled to both Gs and Gi, whereas the beta3a-AR coupled solely to Gs. 3. Examination of four splice variants of the human alpha1A-AR showed that all were able to couple to pertussis toxin-sensitive G-proteins, even though they have radically different C-terminal regions. 4. Comparison of the effects of the beta3-AR ligands CL316243 and SR59230A showed that both can activate the mouse beta3-AR but that SR59230A uses pathways other than cAMP accumulation in 3T3-F442A cells. 5. Examination of a series of alpha1-AR agonists for their ability to activate a number of signalling pathways revealed that A61603 acted as a full agonist in all assays, whereas oxymetazoline was unable to cause cAMP accumulation, suggesting agonist-selective signalling at the human alpha1A-AR.

The HIV-1 nucleoside reverse transcriptase inhibitors stavudine and zidovudine alter adipocyte functions in vitro

Nucleoside analogues are suspected of playing a role in peripheral fat loss in patients during long-term treatment with antiretroviral drugs. We compared the long-term effects of stavudine (10 microM), zidovudine (1 muM), didanosine (10 microM), abacavir (4 microM), lamivudine (10 microM), and tenofovir (1 microM), near their maximum concentration values, on the differentiation, lipid accumulation, survival and mitochondrial function of differentiating 3T3-F442A and differentiated 3T3-L1 adipocytes. None of the nucleoside reverse transcriptase inhibitors (NRTI) markedly altered the differentiation of 3T3-F442A cells, as shown by the unmodified percentage of cells with lipid droplets on day 7 and the expression of the early differentiation markers CCAAT/enhancer binding protein (C/EBP) beta (on day 2) and sterol regulatory element-binding protein. However, stavudine and zidovudine altered the lipid phenotype, decreasing the lipid content and expression of markers involved in lipid metabolism, namely C/EBPalpha, peroxisome proliferator-activated receptor gamma, adipocyte lipid binding protein 2, fatty acid synthase and acetyl-coenzyme A carboxylase. Stavudine and zidovudine, contrary to the other NRTI, drove 5-10% of 3T3-F442A cells towards apoptosis, and reduced the lipid content and survival of differentiated 3T3-L1 adipocytes. Stavudine and zidovudine increased mitochondrial mass by two to fourfold, and lowered the mitochondrial membrane potential (JC-1 stain) as did zalcitabine (0.2 microM). Co-treatment with zidovudine plus lamivudine, or zidovudine plus lamivudine and abacavir, did not increase the effect of zidovudine on cell viability or apoptosis. The thymidine analogues stavudine and zidovudine decreased lipid content, mitochondrial activity, and adipocyte survival in vitro.

Murine glypican-4 gene structure and expressionSp1 and Sp3 play a major role in glypican-4 expression in 3T3-F442A cells

Murine glypican-4 gene structure and expressionSp1 and Sp3 play a major role in glypican-4 expression in 3T3-F442A cells

Antiretroviral Drugs with Adverse Effects on Adipocyte Lipid Metabolism and Survival Alter the Expression and Secretion of Proinflammatory Cytokines and AdiponectinIn Vitro

ObjectiveThe lipodystrophy syndrome is a major adverse effect of highly active antiretroviral therapy (HAART), associated with altered circulating levels and adipose tissue mRNA expression of proinflammatory cytokines interleukin-6 (IL-6) and tumour necrosis factor (TNF)α, and adiponectin. Proinflammatory cytokines and adiponectin, which are secreted by adipose tissue, regulate fat metabolism, insulin sensitivity and adipose cell apoptosis. We examined the direct effects of individual antiretrovirals on lipid metabolism and cytokine and adiponectin production by cultured adipocytes.MethodsDifferentiating 3T3-F442A cells and differentiated 3T3-L1 adipocytes were treated for 12 or 4 days, respectively, with protease inhibitors (PIs) indinavir, nelfinavir, amprenavir, lopinavir and ritonavir, or nucleoside reverse transcriptase inhibitors (NRTIs) stavudine and zidovudine, at near-Cmaxconcentrations. Lipid metabolism was estimated by Oil Red O staining of intracellular lipids, mRNA expression of fatty acid synthase and adipocyte lipid binding protein 2, and insulin activation of lipogenesis. Apoptosis was estimated by flow cytometry. The expression and secretion of proinflammatory cytokines (IL-6, TNFα and IL-1β) and adiponectin were evaluated by real-time reverse transcription PCR and ELISA.ResultsChronic treatment of 3T3-F442A differentiating adipocytes and differentiated 3T3-L1 adipocytes with PIs and NRTIs reduced lipid accumulation, mRNA expression of lipid markers and insulin-induced lipogenesis. IL-6, TNFα, IL-1β and adiponectin expression and secretion were markedly altered in differentiating 3T3-F442A adipocytes. PIs had either no effect on differentiated 3T3-L1 adipocytes (TNFα expression and secretion) or their effect was less marked than in 3T3-F442A cells. Indinavir and amprenavir did not alter cytokine secretion and expression by mature adipocytes. The effects of stavudine and zidovudine on differentiating and mature adipocytes were similar, despite the difference in treatment procedure. The drugs with the strongest effect on TNFα expression also increased adipocyte apoptosis, in contrast to the drugs that only moderately increased TNFα expression.ConclusionsThese results suggest that increased cytokine and decreased adiponectin secretion and expression induced by some PIs and NRTIs may contribute to the adipose tissue loss (via apoptosis and lipid leakage) and insulin resistance associated with the lipodystrophy syndrome.

Regulation of adipocyte differentiation and insulin action with rapamycin

Here, we demonstrated that inhibition of mTOR with rapamycin has negative effects on adipocyte differentiation and insulin signaling. Rapamycin significantly reduced expression of most adipocyte marker genes including PPARγ, adipsin, aP2, ADD1/SREBP1c, and FAS, and decreased intracellular lipid accumulation in 3T3-L1 and 3T3-F442A cells, suggesting that rapamycin would affect both lipogenesis and adipogenesis. Contrary to the previous report that suppressive effect of rapamycin on adipogenesis is limited to the clonal expansion, we revealed that its inhibitory effect persisted throughout the process of adipocyte differentiation. Thus, it is likely that constitutive activation of mTOR might be required for the execution of adipogenic programming. In differentiated 3T3-L1 adipocytes, chronic treatment of rapamycin blunted the phosphorylation of AKT and GSK, which is stimulated by insulin, and reduced insulin-dependent glucose uptake activity. Taken together, these results suggest that rapamycin not only prevents adipocyte differentiation by decrease of adipogenesis and lipogenesis but also downregulates insulin action in adipocytes, implying that mTOR would play important roles in adipogenesis and insulin action.

Murine glypican-4 gene structure and expression: Sp1 and Sp3 play a major role in glypican-4 expression in 3T3-F442A cells

In this report we describe the genomic organization of the mouse glypican-4 (Gpc4), an analysis of its promoter and its transcriptional regulation in the 3T3-F442A adipocyte cell line. The Gpc4 gene consists of nine exons separated by eight introns. A series of deletion mutants and 4391 bp of the 5′-flanking region were cloned into pGL3-BASIC upstream of the luciferase reporter gene and transfected into 3T3-F442A adipocytes. Analysis of a 4.3-kb DNA fragment at the 5′-flanking region of this gene revealed that the Gpc4 promoter is a TATA-less promoter with a large cluster of GC boxes. Competitive electrophoretic mobility shift and supershift assays identified a cluster of nine functional GC boxes binding Sp1 and Sp3 in this region. Transactivation experiments in insect cells showed that both Sp1 and Sp3 are major activators of the Gpc4 promoter. Gpc4 is expressed in adipocytes where its expression is highest in confluent 3T3-F442A adipoblasts and decreases dramatically as cells differentiate. Sp protein analyses demonstrated a major decrease in Sp3 protein in differentiated adipocytes as compared to undifferentiated adipoblasts. These experiments show that Gpc4 is developmentally regulated in 3T3-F442A adipocytes and suggest that Sp transcription factors play a significant role in the regulated expression of Gpc4.

Caveolar and Lipid Raft Localization of the Growth Hormone Receptor and Its Signaling Elements: IMPACT ON GROWTH HORMONE SIGNALING

The growth hormone receptor (GHR) is a cell surface receptor that mediates the somatogenic and metabolic effects of the growth hormone (GH). GHR signaling is transduced via the receptor-associated cytoplasmic tyrosine kinase called Janus protein kinase 2 (JAK2). The major intracellular signaling systems activated by JAK2 in response to GH include the signal transducer and activator of transcription (STAT) 5 and extracellular signal-regulated kinase (ERK)-1 and -2 pathways. In this report, we investigate the role of cholesterol-rich plasma membrane microdomains (caveolae and lipid rafts) in GH signaling. By subcellular fractionation of the GH-responsive 3T3-F442A murine preadipocyte, we found dramatic enrichment (6.7-fold) of plasma membrane GHR in the caveolae membranes (CM). JAK2 was also represented in the CM fraction, but was less enriched (2.5-fold) than GHR. ERK1/2 and the important ERK pathway upstream small adaptor protein, Grb2 (growth factor receptor-bound protein 2), were also enriched in caveolae (2.3- and 8.3-fold, respectively), but STAT5 was barely detected in the same fraction. Correspondingly, GH-induced tyrosine-phosphorylated GHR, JAK2, and ERK1/2 were highly represented in the CM fraction, whereas tyrosine-phosphorylated STAT5 was enriched in the non-membranous fraction of the post-nuclear supernatant. Additionally, GH induced further accumulation of GHR, Grb2, and SHC proteins in the CM fraction. Interestingly, treatment of the cells with the caveolae-disrupting agent, methyl-beta-cyclodextrin (mbetaCD), selectively inhibited GH-induced ERK1/2 activation but not STAT5 phosphorylation; repletion of cholesterol in mbetaCD-treated cells restored GH-induced ERK activation. Comparison of 3T3-F442A cells with the GHR-expressing human IM-9 lymphoblasts revealed similar enrichment of GHR in the lipid raft fraction of IM-9 as in the CM fraction of 3T3-F442A, but there were dramatic differences in the ERKs and Grb2. The IM-9 cell, in which ERKs are not activated by GH, displayed no enrichment of ERKs and Grb2 in the lipid raft fraction. Our results suggest that localization of GHRs in the CM fraction of the plasma membrane plays important roles in signaling.

Growth hormone alters epidermal growth factor receptor binding affinity via activation of extracellular signal-regulated kinases in 3T3-F442A cells.

Epidermal growth factor receptor (EGFR) is a transmembrane protein that binds EGF in its extracellular domain and initiates signaling via intrinsic tyrosine kinase activity in its cytoplasmic domain. EGFR is important in development, cellular proliferation, and cancer. GH is a critical growthpromoting and metabolic regulatory hormone that binds the GH receptor, thereby engaging various signaling pathways, including ERKs. Prior studies suggest cross-talk between the GH receptor and EGFR signaling systems. Using the GH- and EGF-responsive 3T3-F442A preadipocyte, we previously observed that GH, in addition to causing EGFR tyrosine phosphorylation, also induced EGFR phosphorylation that was detected by PTP101, an antibody reactive with ERK consensus phosphorylation sites. This latter phosphorylation was prevented by pretreatment with MAPK kinase (MEK)1 inhibitors, suggesting ERK pathway dependence. Furthermore, GH cotreatment with EGF markedly slowed EGF-induced EGFR degradation and down-regulation, thereby potentiating EGF-induced EGFR signaling. These effects were also MEK1 dependent and suggested ERK pathway-dependent influence of GH on EGF-induced EGFR postendocytic trafficking and signaling. We now explore the impact of GH on cell surface binding of EGF in 3T3-F442A cells. We found that GH pretreatment caused transient, but substantial, lessening of (125)I-EGF binding. Competitive binding experiments revealed that the decreased binding was primarily due to decreased affinity, rather than a change in the number of EGF binding sites. The effect of GH on EGF binding was concentration dependent and temporally correlated with GH-induced ERK activation and EGFR PTP101-reactive phosphorylation. Blockade of the MEK1/ERK but not the protein kinase C pathway, prevented GH's effects on EGF binding, and our results indicate that the mechanisms of GH- and phorbol-12-myristate-13-acetateinduced inhibition of EGF binding differ substantially. Overall, our findings suggest that GH can modulate both EGF binding kinetics and the EGFR's postbinding signaling itinerary in a MEK1/ERK pathway-dependent fashion.

Physical and functional interaction of growth hormone and insulin-like growth factor-I signaling elements.

GH and IGF-I are critical regulators of growth and metabolism. GH interacts with the GH receptor (GHR), a cytokine superfamily receptor, to activate the cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), and initiate intracellular signaling cascades. IGF-I, produced in part in response to GH, binds to the heterotetrameric IGF-I receptor (IGF-IR), which is an intrinsic tyrosine kinase growth factor receptor that triggers proliferation, antiapoptosis, and other biological actions. Previous in vitro and overexpression studies have suggested that JAKs may interact with IGF-IR and that IGF-I stimulation may activate JAKs. In this study, we explore interactions between GHR-JAK2 and IGF-IR signaling pathway elements utilizing the GH and IGF-I-responsive 3T3-F442A and 3T3-L1 preadipocyte cell lines, which endogenously express both the GHR and IGF-IR. We find that GH induces formation of a complex that includes GHR, JAK2, and IGF-IR in these preadipocytes. The assembly of this complex in intact cells is rapid, GH concentration dependent, and can be prevented by a GH antagonist, G120K. However, it is not inhibited by the kinase inhibitor, staurosporine, which markedly inhibits GHR tyrosine phosphorylation. Moreover, complex formation does not appear dependent on GH-induced activation of the ERK or phosphatidylinositol 3-kinase signaling pathways or on the tyrosine phosphorylation of GHR, JAK2, or IGF-IR. These results suggest that GH-induced formation of the GHR-JAK2-IGF-IR complex is governed instead by GH-dependent conformational change(s) in the GHR and/or JAK2. We further demonstrate that GH and IGF-I can synergize in acute aspects of signaling and that IGF-I enhances GH-induced assembly of conformationally active GHRs. These findings suggest the existence of previously unappreciated relationships between these two hormones.

Histamine does not induce an amine oxidase-dependent adipogenic effect in murine preadipocytes.

Mice deficient in H3-receptors exhibit increased food intake, body weight, and adiposity [1]. Also intracerebroventricular administration of histamine suppresses appetite [2]. Chronic administration of H3-receptor antagonists reduces food intake and body weight gain in rodents. Although changes in release of neurotransmitters which regulate appetite may explain observed changes in food consumption and subsequent alterations of fat deposition, there may be a direct action of histamine on adipose cells. Previously we reported that histamine stimulates glucose transport in rat adipocytes, in presence of vanadate [3]. Also histamine has a dual influence on lipid mobilization: it stimulates lipolysis when H1receptors and amine oxidases are blocked [4] or inhibits glycerol release when incubated at high concentration with mature adipocytes [5]. Here, we studied whether histamine was able to influence adipose differentiation of cultured preadipocytes. Since amine oxidation by semicarbazide-sensitive amine oxidase (SSAO) has been shown to mediate adipogenic effect of benzylamine [6] or methylamine [7] in 3T3-F442A cells, we also examined whether histamine was subjected to oxidative deamination in these murine cells.

A simple and sensitive assay for GH activity based on 3T3-F442A cell differentiation

We describe a fast, sensitive, specific, and simple in vitro assay for GH biological activity, based on the differentiation of 3T3-F442A cells into adipocytes. The 3T3-F442A cells were directly plated at 1.5 × 10 4 cells/cm 2 in medium with or without various concentrations of human growth hormone (hGH). After 7 days, cells were lysed with buffer containing 0.5 % (v/v) Triton X-100, and adipose conversion was quantitated by the activity of the adipogenic enzyme glycerophosphate dehydrogenase. The assay is highly sensitive and specific for GH from different species. These culture conditions have shortened the time for the cells to undergo adipose differentiation, and they might also be useful to design and test drugs or agents that modify adipocyte differentiation or lipid metabolism, or for evaluation of cytotoxic and pharmacologic effects of drugs and other compounds.

Differential effect of HIV protease inhibitors on adipogenesis: intracellular ritonavir is not sufficient to inhibit differentiation.

Lipodystrophy is a major side effect of HIV protease inhibitor (PI) antiretroviral therapy. It has been shown that protease inhibitors interfere in vitro with adipocyte differentiation. However, there is no evidence that PIs accumulate into preadipocytes and adipocytes and that intra-cellular accumulation is sufficient to alter differentiation. We assessed the effect of six different PIs on the differentiation of cells from four clonal lines. We also studied the capacity of ritonavir to accumulate both into drug-sensitive and drug-resistant cultured adipocytes. Adipocyte differentiation of mouse 3T3-F442A, 3T3-L1 and Ob1771 cells as well as embryonic stem cells were investigated at pharmacological concentrations of indinavir, saquinavir, ritonavir, amprenavir, nelfinavir and lopinavir. We used a sensitive ELISA to determine intracellular concentration of ritonavir from 3T3-L1 and Ob1771 preadipocytes. Nelfinavir and lopinavir inhibited adipocyte differentiation whereas amprenavir was ineffective. Indinavir, saquinavir and ritonavir inhibited differentiation of 3T3-L1 and 3T3-F442A cells but did not alter differentiation of either Ob1771 or embryonic stem cells. We showed that ritonavir accumulated in preadipocytes and fully differentiated 3T3-L1 adipocytes as a function of its extracellular concentration. Although Ob1771 cells were resistant and 3T3-L1 cells were sensitive to ritonavir, the drug accumulated to similar levels in both cases. Protease inhibitors inhibit adipocyte differentiation depending on the cell model used. We showed for the first time that ritonavir accumulates into preadipocytes and adipocytes, suggesting a direct effect on intracellular targets. However, intracellular accumulation was clearly not sufficient as Ob1771 cells remained resistant to the inhibitory effect of ritonavir.

Subnuclear Localization of C/EBPβ Is Regulated by Growth Hormone and Dependent on MAPK

Localization of transcription regulatory proteins in the nucleus is dynamically regulated, and may alter nucleoplasmic concentrations and/or assembly of multimolecular transcription regulatory complexes, which ultimately regulate gene expression. Since growth hormone (GH) regulates multiple transcription factors including C/EBP beta, the effect of GH on the subcellular localization of C/EBP beta was examined in 3T3-F442A preadipocytes. Indirect immunofluorescence shows that C/EBP beta is diffusely distributed in nuclei of quiescent cells. Within 5 min of GH treatment, the diffuse pattern dramatically becomes punctate. The relocalization of C/EBP beta coincides with DAPI staining of heterochromatin. Further, C/EBP beta and heterochromatin protein (HP)-1 alpha colocalize in the nucleus, consistent with localization of C/EBP beta to pericentromeric heterochromatin. In contrast, C/EBP delta exhibits a diffuse distribution in the nucleus that is not modified by GH treatment. C/EBP beta is rapidly and transiently phosphorylated on a conserved MAPK consensus site in response to GH (Piwien-Pilipuk, G., MacDougald, O. A., and Schwartz, J. (2002) J. Biol. Chem. 277, 44557-44565). Indirect immunofluorescence using antibodies specific for C/EBP beta phosphorylated on the conserved MAPK site shows that GH also rapidly induces a punctate pattern of staining for the phosphorylated C/EBP beta. In addition, phosphorylated C/EBP beta colocalizes to pericentromeric heterochromatin. The satellite DNA present in heterochromatin contains multiple C/EBP binding sites. DNA binding analysis shows that C/EBP beta, C/EBP delta, and C/EBP alpha (p42 and p30 forms) can bind to satellite DNA as homo- or heterocomplexes in vitro. Importantly, GH rapidly and transiently increases binding of endogenous C/EBP beta from 3T3-F442A cells to satellite DNA. Further, the GH-promoted nuclear relocalization of C/EBP beta to pericentromeric heterochromatin was prevented by the MEK inhibitor U0126. This observation suggests that GH-dependent MAPK activation plays a role in the regulation of nuclear relocalization of C/EBP beta. Nuclear redistribution introduces a new level of transcriptional regulation in GH action, since GH-mediated phosphorylation and nuclear redistribution of C/EBP beta may be coordinated to achieve spatial-temporal control of gene expression.

Protein kinase C-epsilon promotes adipogenic commitment and is essential for terminal differentiation of 3T3-F442A preadipocytes.

The role of protein kinase C (PKC) isoforms in the commitment of multipotent fibroblasts to the adipocyte lineage and in their terminal differentiation into mature adipocytes was investigated. Ectopic overexpression of PKC-epsilon, but not other PKC isoforms, committed multipotent NIH-3T3 cells to adipogenic differentiation in the presence of hormonal inducers. In committed 3T3-F442A preadipocytes, PKC-epsilon protein expression increased during the course of terminal differentiation and cell-permeable PKC-epsilon inhibitory peptides, which prevent interaction with RACK (receptor for activated C-kinase) proteins, severely inhibited differentiation. PKC-epsilon accumulated in the nuclei of 3T3-F442A cells shortly after induction of differentiation and exhibited a distinctive punctate speckling immunocytochemical staining pattern. The spatiotemporal aspects of PKC-epsilon localization and expression coincided with that of C/EBP-beta, a transcription factor critically involved in promoting the early phase of adipogenesis. Collectively, these results demonstrate a role for PKC-epsilon in both adipogenic commitment and preadipocyte terminal differentiation.