Interleukin-15 stimulates adiponectin secretion by 3T3-L1 adipocytes: evidence for a skeletal muscle-to-fat signaling pathway

Interleukin-15 (IL-15) is a cytokine which is highly expressed in skeletal muscle tissue, and which has anabolic effects on skeletal muscle protein dynamics both in vivo and in vitro. Additionally, administration of IL-15 to rats and mice inhibits white adipose tissue deposition. To determine if the action of IL-15 on adipose tissue is direct, the capacity of cultured murine 3T3-L1 preadipocytes and adipocytes to respond to IL-15 was examined. IL-15 administration inhibited lipid accumulation in differentiating 3T3-L1 preadipocytes, and stimulated secretion of the adipocyte-specific hormone adiponectin by differentiated 3T3-L1 adipocytes. The latter observation constitutes the first report of a cytokine or growth factor which stimulates adiponectin production. IL-15 mRNA expression by cultured 3T3-L1 adipogenic cells and C2C12 murine skeletal myogenic cells was also examined. Quantitative real-time PCR indicated IL-15 mRNA was expressed by C2C12 skeletal myogenic cells, and was upregulated more than 10-fold in differentiated skeletal myotubes compared to undifferentiated myoblasts. In contrast, 3T3-L1 cells expressed little or no IL-15 mRNA at either the undifferentiated preadipocyte or differentiated adipocyte stages. These findings provide support for the hypothesis that IL-15 functions in a muscle-to-fat endocrine axis which modulates fat:lean body composition and insulin sensitivity.     

Characterization and binding properties of human fetal lung fatty acid-binding proteins

When delipidated Mr>10,000 cut-off human fetal lung cytosol was separated on gel filtration and ion-exchange chromatography on Auto-FPLC system, two fatty acid-binding proteins (FABPs) of pI 6.9 and pI 5.4 were purified to homogeneity. On Western blotting analysis with the anti-human fetal lung pI 6.9 FABP, these two proteins showed immunochemical cross reactivity with each other and with purified hepatic FABPs but not with cardiac or gut FABP. These two FABPs have identical molecular mass of 15.2 kDa, which is slightly higher than that of the hepatic proteins (14.2 kDa). Carbohydrate covalently linked to FABPs, that may substantially add to the molecular mass, was not detected in the purified protein preparations. Amino acid analysis revealed that both the proteins have same amino acid composition each containing one Trp residue that is lacking in hepatic FABP. Different isoforms of lung FABP exhibited different binding ability for their natural ligands. These proteins bind palmitoyl CoA with higher affinity than oleic acid. pI 6.9 FABP can more rapidly and efficiently transfer fatty acid than can pI 5.4 FABP from unilammelar liposomes. Thus these FABPs may play a critical role in fatty acid transport during human fetal lung development.Abbreviations AO anthroyloxy - 12-AS 12-(9-anthroyloxy)stearic acid - FABP fatty acid-binding protein - NBD-PE [N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine - Pal-CoA palmitoyl coenzyme A - PITC phenylisothiocyanate - PBS phosphate-buffered saline - PtdCho phosphatidylcholine - SUV small unilamellar vesicle - Tris tris(hydroxymethyl) amino methane     

Phosphotyrosyl turnover in insulin signaling. Characterization of two membrane-bound pp15 protein tyrosine phosphatases from 3T3-L1 adipocytes.

It was shown previously that 422 (aP2) protein, a 15-kDa fatty acid binding protein, is phosphorylated on Tyr19 both in vitro by the insulin receptor tyrosine kinase and in intact 3T3-L1 adipocytes treated with insulin and phenylarsine oxide (PAO). Phospho-422(aP2) protein (pp15) accumulates in cells treated with insulin and PAO because the arsenical blocks turnover of the phosphoryl group of pp15. These findings suggest that a PAO-sensitive enzyme mediates turnover of the pp15 tyrosine phosphoryl group. We have purified and characterized two membrane protein tyrosine phosphatases (PTPases) from 3T3-L1 adipocytes that catalyze hydrolysis of phospho-Tyr19 of authentic pp15. These enzymes, designated PTPases HA1 and HA2, were purified approximately 20,000-fold and approximately 15,000-fold, respectively, and shown to differ markedly in their sensitivity to both vanadate and phosphotyrosine. Both enzymes are inhibited by PAO and accordingly can be labeled with 4-[125I]iodo-PAO. By this method, it was demonstrated that PTPases HA1 and HA2 have molecular masses of approximately 60 kDa and approximately 38 kDa, respectively. Both enzymes exhibit substrate preference for pp15 when compared with other phosphotyrosine-containing protein substrates. Proteins containing phosphoserine and phosphothreonine do not serve as substrates for the enzymes. The pp15 PTPase HA2 is expressed both in 3T3-L1 preadipocytes and adipocytes, whereas pp15 PTPase HA1 is expressed only in 3T3-L1 adipocytes.     

Induction of adipose fatty acid binding protein (a-FABP) by insulin-like growth factor-1 (IGF-1) in 3T3-L1 preadipocytes.

Murine 3T3-L1 cells were cultured in the presence of fetal bovine serum (FBS) washed with an anion exchange resin and charcoal. Using the abundance of a-FABP and fatty acid synthase (FAS) as criteria of differentiation, IGF-1 was found to be 10-fold more potent than insulin as an inducer of preadipocyte differentiation. As little as 0.5 nM IGF-1 induced expression of FAS and a-FABP mRNAs whereas a minimum of 5 nM insulin was required. The data indicate IGF-1 specifically induces the expression of a-FABP in 3T3-L1 preadipocytes whereas the effect of insulin is likely via insulin's binding to the IGF-1 receptor.     

STAT5 activators modulate acyl CoA oxidase (AOX) expression in adipocytes and STAT5A binds to the AOX promoter in vitro

Growth hormone (GH) diminishes adipose tissue mass in vivo and prolactin (PRL) can also modulate adipocyte metabolism. Both GH and PRL are potent activators of STAT5 and exert a variety of effects on adipocyte gene expression. In this study, we have demonstrated that GH and PRL increase the mRNA of acyl CoA oxidase in 3T3-L1 adipocytes. We also identified seven putative STAT elements in the murine AOX promoter. We observed that GH modulates protein binding to the majority of these promoter elements. However, GH induced very potent binding to -1841 to -1825 of the murine AOX promoter. EMSA supershift analysis revealed that this site was specifically bound by STAT5A, but not by STAT1 or STAT3. Taken together, these data strongly suggest that GH directly induces the expression of AOX in adipocytes through STAT5A binding to the -1841 to -1825 site within the AOX promoter. Our observations are consistent with other studies that demonstrate that STAT5 activators modulate fatty acid oxidation.     

A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation

A 12.5-kDa cysteine-rich adipose tissue-specific secretory factor (ADSF/resistin) is a novel secreted protein rich in serine and cysteine residues with a unique cysteine repeat motif of CX(12)CX(8)CXCX(3)CX(10)CXCXCX(9)CC. A single 0.8-kilobase mRNA coding for this protein was found in various murine white adipose tissues including inguinal and epididymal fats and also in brown adipose tissue but not in any other tissues examined. Two species of mRNAs with sizes of 1.4 and 0.8 kilobases were found in rat adipose tissue. Sequence analysis indicates that this is because of two polyadenylation signals, the proximal one with the sequence AATACA with a single base mismatch from murine AATAAA and the distal consensus sequence AATAAA. The mRNA level was markedly increased during 3T3-L1 and primary preadipocyte differentiation into adipocytes. Its expression in adipose tissue is under tight nutritional and hormonal regulation; the mRNA level was very low during fasting and increased 25-fold when fasted mice were refed a high carbohydrate diet. It was also very low in adipose tissue of streptozotocin-diabetes and increased 23-fold upon insulin administration. Upon treatment with the conditioned medium from COS cells transfected with the expression vector, conversion of 3T3-L1 cells to adipocytes was inhibited by 80%. The regulated expression pattern suggesting this factor as an adipose sensor for the nutritional state of the animals and the inhibitory effect on adipocyte differentiation implicate its function as a feedback regulator of adipogenesis.     

Heart-type fatty acid-binding protein is essential for efficient brown adipose tissue fatty acid oxidation and cold tolerance

Brown adipose tissue has a central role in thermogenesis to maintain body temperature through energy dissipation in small mammals and has recently been verified to function in adult humans as well. Here, we demonstrate that the heart-type fatty acid-binding protein, FABP3, is essential for cold tolerance and efficient fatty acid oxidation in mouse brown adipose tissue, despite the abundant expression of adipose-type fatty acid-binding protein, FABP4 (also known as aP2). Fabp3(-/-) mice exhibit extreme cold sensitivity despite induction of uncoupling and oxidative genes and hydrolysis of brown adipose tissue lipid stores. However, using FABP3 gain- and loss-of-function approaches in brown adipocytes, we detected a correlation between FABP3 levels and the utilization of exogenous fatty acids. Thus, Fabp3(-/-) brown adipocytes fail to oxidize exogenously supplied fatty acids, whereas enhanced Fabp3 expression promotes more efficient oxidation. These results suggest that FABP3 levels are a determinant of fatty acid oxidation efficiency by brown adipose tissue and that FABP3 represents a potential target for modulation of energy dissipation.     

Adipose tissue gene expression of factors related to lipid processing in obesity

Background

Adipose tissue lipid storage and processing capacity can be a key factor for obesity-related metabolic disorders such as insulin resistance and diabetes. Lipid uptake is the first step to adipose tissue lipid storage. The aim of this study was to analyze the gene expression of factors involved in lipid uptake and processing in subcutaneous (SAT) and visceral (VAT) adipose tissue according to body mass index (BMI) and the degree of insulin resistance (IR).

Methods and Principal Findings

VLDL receptor (VLDLR), lipoprotein lipase (LPL), acylation stimulating protein (ASP), LDL receptor-related protein 1 (LRP1) and fatty acid binding protein 4 (FABP4) gene expression was measured in VAT and SAT from 28 morbidly obese patients with Type 2 Diabetes Mellitus (T2DM) or high IR, 10 morbidly obese patients with low IR, 10 obese patients with low IR and 12 lean healthy controls. LPL, FABP4, LRP1 and ASP expression in VAT was higher in lean controls. In SAT, LPL and FABP4 expression were also higher in lean controls. BMI, plasma insulin levels and HOMA-IR correlated negatively with LPL expression in both VAT and SAT as well as with FABP4 expression in VAT. FABP4 gene expression in SAT correlated inversely with BMI and HOMA-IR. However, multiple regression analysis showed that BMI was the main variable contributing to LPL and FABP4 gene expression in both VAT and SAT.

Conclusions

Morbidly obese patients have a lower gene expression of factors related with lipid uptake and processing in comparison with healthy lean persons.     

TSH signaling and cell survival in 3T3-L1 preadipocytes

Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29-76% in serum-deprived (6 h) preadipocytes treated with 1-20 microM TSH. In the presence of 20 microM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.     

Tissue specific expression of p422 protein, a putative lipid carrier, in mouse adipocytes

The differentiation of 3T3-L1 preadipocytes leads to the expression of a new protein, p422, and its mRNA. This protein has 70% and 20-30% amino acid sequence homology to myelin P2 and the fatty acid binding proteins of liver and intestine, respectively. Investigation of the distribution in mouse tissues of p422 protein by immunoblotting and of p422 mRNA by cDNA hybridization indicates that they are expressed only in adipose tissue. Liver and intestinal fatty acid binding protein mRNA's were not detectable in mouse adipose tissue or in 3T3-L1 adipocytes. It is suggested that p422 functions as an adipocyte fatty acid binding protein.     

Amino acid sequence and some ligand binding properties of fatty acid-binding protein from bovine brain

Summary A fatty acid-binding protein (FABP) from the cytosol of bovine brain was purified by Sephadex G-75 filtration and electrofocusing. The purified protein migrated as a single protein band in 15% polyacrylamide gel electrophoresis with an apparent molecular mass of 14.7 kDa. To ascertain that the purified protein was a FABP, it was submitted to fatty acid-binding tests. Oleic and palmitic acids bound to brain FABP but this was not the case for palmitoyl CoA. By Scatchard analysis the ligand binding values were: Kd = 0.28 µM, Bmax (mol/mol) = 0.6 for oleic acid and Kd = 0.8 µM, Bmax (mol/mol) = 2.1 for palmitic acid. The complete amino acid sequence of the brain FABP was determined and a microheterogeneity was observed. Sequence comparison with other FABPs of known sequence and the observed microheterogeneity demonstrated the presence in brain of several homologous FABPs closely related to heart FABP.This paper corresponds to a communication at the first international workshop on fatty acid binding proteins (Maastricht, the Netherlands, September 4–5, 1989).     

Modulation of adipogenesis-related gene expression by estrogen-related receptor gamma during adipocytic differentiation

Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in oxidative metabolism and mitochondrial biogenesis in brown adipose tissue and heart. However, the physiological role of ERRgamma in adipogenesis and the development of white adipose tissue has not been well studied. Here we show that ERRgamma was up-regulated in murine mesenchyme-derived cells, especially in ST2 and C3H10T1/2 cells, at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. The up-regulation of ERRgamma mRNA was also observed in inguinal white adipose and brown adipose tissues of mice fed a high-fat diet. Gene knockdown by ERRgamma-specific siRNA results in mRNA down-regulation of adipogenic marker genes including fatty acid binding protein 4, PPARgamma, and PGC-1beta in a preadipocyte cell line 3T3-L1 preadipocytes and mesenchymal ST2 and C3H10T1/2 cells in the adipogenesis medium. In contrast, stable expression of ERRgamma in 3T3-L1 cells resulted in up-regulation of these adipogenic marker genes under the adipogenic condition. These results suggest that ERRgamma positively regulate the adipocyte differentiation with modulating the expression of various adipogenesis-related genes.     

Galectin-12, an Adipose-expressed Galectin-like Molecule Possessing Apoptosis-inducing Activity.

Galectins constitute a family of proteins that bind to beta-galactoside residues and have diverse physiological functions. Here we report on the identification of a galectin-like molecule, galectin-12, in a human adipose tissue cDNA library. The protein contained two potential carbohydrate-recognition domains with the second carbohydrate-recognition domain being less conserved compared with other galectins. In vitro translated galectin-12 bound to a lactosyl-agarose column far less efficiently than galectin-8. Galectin-12 mRNA was predominantly expressed in adipose tissue of human and mouse and in differentiated 3T3-L1 adipocytes. Caloric restriction and treatment of obese animals with troglitazone increased galectin-12 mRNA levels and decreased the average size of the cells in adipose tissue. The induction of galectin-12 expression by the thiazolidinedione, troglitazone, was paralleled by an increase in the number of apoptotic cells in adipose tissue. Immunocytochemical analysis revealed that galectin-12 was localized in the nucleus of adipocytes, and transfection with galectin-12 cDNA induced apoptosis of COS-1 cells. These results suggest that galectin-12, an adipose-expressed galectin-like molecule, may participate in the apoptosis of adipocytes.     

NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes

The nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway responds to oxidative stress via control of several antioxidant defense gene expressions. Recent efforts demonstrate that Nrf2 modulates development of adiposity and adipogenesis. One of the major Nrf2-regulated proteins, NAD(P)H:quinone oxidoreductase 1 (NQO1), is implicated in the development of adipose tissue and obesity. However, little is known about in situ disposition of Nrf2, Keap1, and NQO1 during adipogenesis in isolated adipocytes. Based on literature data, we hypothesized that adipocyte differentiation would increase expression of the Nrf2/Keap1 pathway and NQO1. Using murine 3T3-L1 preadipocytes, we mapped an increase in NQO1 protein at limited clonal expansion and postmitotic growth arrest (Days 1-3) stages and a decrease in terminally differentiated (Day 8) adipocytes that lasted for several days afterward. Conversely, NQO1, Nrf2, and Keap1 mRNA expressions were all increased in differentiated adipocytes (Days 11-14), indicating a discrepancy between steady-state mRNA levels and resulting protein. Treatment of differentiated 3T3-L1 adipocytes with glycogen synthase kinase-3β (GSK-3β) inhibitor, LiCl, led to 1.9-fold increase in NQO1 protein. Sulforaphane enhanced NQO1 protein (10.5-fold) and blunted triglyceride and FABP4 accumulation. The decrement in triglyceride content was partially reversed when NQO1 activity was pharmacologically inhibited. These data demonstrate a biphasic response of Nrf2 and NQO1 during adipocyte differentiation that is regulated by Keap1- and GSK-3β-dependent mechanisms, and that hypertrophy is negatively regulated by NQO1 activity.     

Small-molecule inhibitors of FABP4/5 ameliorate dyslipidemia but not insulin resistance in mice with diet-induced obesity

Fatty acid binding protein-4 (FABP4) and FABP5 are two closely related FA binding proteins expressed primarily in adipose tissue and/or macrophages. The small-molecule FABP4 inhibitor BMS309403 was previously reported to improve insulin sensitivity in leptin-deficient Lep(ob)/Lep(ob) (ob/ob) mice. However, this compound was not extensively characterized in the more physiologically relevant animal model of mice with diet-induced obesity (DIO). Here, we report the discovery and characterization of a novel series of FABP4/5 dual inhibitors represented by Compounds 1-3. Compared with BMS309403, the compounds had significant in vitro potency toward both FABP4 and FABP5. In cell-based assays, Compounds 2 and 3 were more potent than BMS309403 to inhibit lipolysis in 3T3-L1 adipocytes and in primary human adipocytes. They also inhibited MCP-1 release from THP-1 macrophages as well as from primary human macrophages. When chronically administered to DIO mice, BMS309403 and Compound 3 reduced plasma triglyceride and free FA levels. Compound 3 reduced plasma free FAs at a lower dose level than BMS309403. However, no significant change was observed in insulin, glucose, or glucose tolerance. Our results indicate that the FABP4/5 inhibitors ameliorate dyslipidemia but not insulin resistance in DIO mice.     

Chemerin--a new adipokine that modulates adipogenesis via its own receptor

Chemerin, an 18 kDa protein secreted by adipose tissue, was reported to modulate immune system function through its binding to the chemerin receptor (chemerinR). We herein demonstrate that chemerin also influences adipose cell function. Our data showed that chemerin and chemerinR mRNA expressions were highly expressed in adipose tissues, and that their expression levels were up-regulated in mice fed a high-fat diet. Both chemerin and chemerinR mRNA expression dramatically increased during the differentiation of 3T3-L1 cells and human preadipocytes into adipocytes. Furthermore, recombinant chemerin induced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2) and lipolysis in differentiated 3T3-L1 adipocytes. Thus, the adipokine chemerin likely regulates adipocyte function by autocrine/paracrine mechanisms.