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Adipose tissue macrophages are associated with insulin resistance and are linked to changes in the extracellular matrix. To better characterize adipose macrophages, the extracellular matrix, and adipocyte-macrophage interactions, gene expression from adipose tissue and the stromal vascular fraction was assessed for markers of inflammation and fibrosis, and macrophages from obese and lean subjects were counted and characterized immunohistochemically. Coculture experiments examined the effects of adipocyte-macrophage interaction. Collagen VI gene expression was associated with insulin sensitivity and CD68 (r = -0.56 and 0.60, P < 0.0001) and with other markers of inflammation and fibrosis. Compared with adipose tissue from lean subjects, adipose tissue from obese subjects contained increased areas of fibrosis, which correlated inversely with insulin sensitivity (r = -0.58, P < 0.02) and positively with macrophage number (r = 0.70, P < 0.01). Although macrophages in crownlike structures (CLS) were more abundant in obese adipose tissue, the majority of macrophages were associated with fibrosis and were not organized in CLS. Macrophages in CLS were predominantly M1, but most other macrophages, particularly those in fibrotic areas, were M2 and also expressed CD150, a marker of M2c macrophages. Coculture of THP-1 macrophages with adipocytes promoted the M2 phenotype, with a lower level of IL-1 expression and a higher ratio of IL-10 to IL-12. Transforming growth factor-[beta] (TGF-[beta]) was more abundant in M2 macrophages and was further increased by coculture with adipocytes. Downstream effectors of TGF-[beta], such as plasminogen activator inhibitor-l, collagen VI, and phosphorylated Smad, were increased in macrophages and adipocytes. Thus adipose tissue of insulin-resistant humans demonstrated increased fibrosis, M2 macrophage abundance, and TGF-[beta] activity. alternatively activated macrophages; inflammation; crownlike structures doi: 10.1152/ajpendo.00329.2010.

Adiponectin, made exclusively by adipocytes, is a 30-kDa secretory protein assembled posttranslationally into low-molecular weight, middle-molecular weight, and high-molecular weight homo-oligomers. PPAR[gamma] ligand thiozolidinediones, which are widely used in the treatment of type II diabetes, increase adiponectin levels. PPAR[gamma] also has several putative ligands that include fatty acid derivatives. Overnight treatment of rat adipocytes with pioglitazone, docosahexaenoic acid (DHA), or eicosapentaenoic acid (EPA) triggered a twofold increase in the synthesis and secretion of HMW adiponectin, and this increase was blocked by the addition of PPAR[gamma] inhibitor GW-9662. Inhibition of glycosylation using 2,2'-dipyridyl decreased the synthesis of high-molecular weight adiponectin by pioglitazone, EPA, and DHA, but there was increased secretion of trimeric adiponectin resulting from increased translation. Although pioglitazone, DHA, and EPA increased adiponectin synthesis by more than 60%, there was no increase in total protein synthesis and no corresponding change in adiponectin mRNA expression, indicating the upregulation of translation. We examined the possibility of transacting factors in the cytoplasmic extracts from adipocytes treated with pioglitazone or DHA. In vitro translation of adiponectin mRNA was inhibited by S-100 fraction of control adipocytes and increased by S-100 extracts from adipocytes treated with pioglitazone or DHA. Consistent with this observation, both pioglitazone and DHA treatments increased the association of adiponectin mRNA with the heavier polysome fractions. Together, these data suggest that pioglitazone and the fish oils DHA or EPA are PPAR[gamma] agonists in adipocytes with regard to adiponectin expression, and the predominant mode of adiponectin stimulation is via an increase in translation. peroxisome proliferator-activated receptor-[gamma]; translational regulation; adipocyte biology; ribonucleic acid-binding proteins; fish oils; thiazolidinediones

Adipose tissue has profound effects on whole-body insulin sensitivity. However, the underlying biological processes are quite complex and likely multifactorial. For instance, the adipose transcriptome is posttranscriptionally modulated by microRNAs, but the relationship between microRNAs and insulin sensitivity in humans remains to be determined. To this end, we utilized an integrative mRNA-microRNA microarray approach to identify putative molecular interactions that regulate the transcriptome in subcutaneous adipose tissue of insulinsensitive (IS) and insulin-resistant (IR) individuals. Using the Nano- String nCounter Human v1 microRNA Expression Assay, we show that 17 microRNAs are differentially expressed in IR vs. IS. Of these, 16 microRNAs (94%) are downregulated in IR vs. IS, including miR-26b, miR-30b, and miR-145. Using Agilent Human Whole Genome arrays, we identified genes that were predicted targets of miR-26b, miR-30b, and miR-145 and were upregulated in IR subjects. This analysis produced ADAM22, MYO5A, LOX, and GM2A as predicted gene targets of these microRNAs. We then validated that miR-145 and miR-30b regulate these mRNAs in differentiated human adipose stem cells. We suggest that use of bioinformatic integration of mRNA and microRNA arrays yields verifiable mRNA-microRNA pairs that are associated with insulin resistance and can be validated in vitro. [ABSTRACT FROM AUTHOR]

Abstract: Objective: A common gain-of-function LPL variant, LPLS447X, has favorable clinical features and involves a C→G base change at nucleotide 1595 of the LPL cDNA, along with a haplotype, which includes other non-coding SNPs. The mechanism for the LPL gain-in-function is not clear. LPL translation is regulated by epinephrine by an RNA–protein complex, consisting of PKA subunits and an A kinase anchoring protein (AKAP), which targets the 3′UTR. Methods: To examine LPL translation of the LPLS447X variant, in vitro translation of LPL mRNA constructs was studied in the presence of cytoplasmic extracts from 3T3-F442A adipocytes treated with/without epinephrine. Results: When the C→G base change at nucleotide 1595 was introduced, LPL mRNA was less susceptible to inhibition by the adipocyte extract. Similarly, a lessened susceptibility to translation inhibition occurred when the complete haplotype was constructed in the full-length 3.6kb LPL mRNA, when an irrelevant coding sequence was introduced into the LPL mRNA construct, and in response to the use of components of the RNA binding complex (PKA C and R subunits, and KH region of AKAP149). Conclusion: These studies suggest that the LPLS447X gain of function may be due to the base change in the LPL mRNA resulting in a decreased susceptibility to translational inhibition. [Copyright &y& Elsevier]

Adiponectin, made exclusively by adipocytes, is a 30-kDa secretory protein assembled posttranslationally into low-molecular weight, middle-molecular weight, and high-molecular weight homo-oligomers. PPAR-γ ligand thiozolidinediones, which are widely used in the treatment of type II diabetes, increase adiponectin levels. PPARγ also has several putative ligands that include fatty acid derivatives. Overnight treatment of rat adipocytes with pioglitazone, docosahexaenoic acid (DHA), or eicosapentaenoic acid (EPA) triggered a twofold increase in the synthesis and secretion of HMW adiponectin, and this increase was blocked by the addition of PPAR-γ inhibitor GW-9662. Inhibition of glycosylation using 2,2'-dipyridyl decreased the synthesis of high-molecular weight adiponectin by pioglitazone, EPA, and DHA, but there was increased secretion of trimeric adiponectin resulting from increased translation. Although pioglitazone, DHA, and EPA increased adiponectin synthesis by more than 60%, there was no increase in total protein synthesis and no corresponding change in adiponectin mRNA expression, indicating the upregulation of translation. We examined the possibility of transacting factors in the cytoplasmic extracts from adipocytes treated with pioglitazone or DHA. In vitro translation of adiponectin mRNA was inhibited by S-100 fraction of control adipocytes and increased by S-100 extracts from adipocytes treated with pioglitazone or DHA. Consistent with this observation, both pioglitazone and DHA treatments increased the association of adiponectin mRNA with the heavier polysome fractions. Together, these data suggest that pioglitazone and the fish oils DHA or EPA are PPAR-γ agonists in adipocytes with regard to adiponectin expression, and the predominant mode of adiponectin stimulation is via an increase in translation. [ABSTRACT FROM AUTHOR]

Adipose lipoprotein lipase (LPL) plays an important role in regulating plasma triglycerides and lipid metabolism. We have previously demonstrated that PKC α depletion using specific antisense oligomers inhibits LPL activity in 3T3 F442A adipocytes. This inhibition of LPL activity was identified as translational and mediated by the interaction of cytoplasmic RNA binding proteins with sequences on the 3′UTR of LPL mRNA. Using in vitro translation experiments we have now identified the minimum essential region on the 3′UTR of LPL mRNA required for the inhibition of translation. These data have been confirmed by RNase protection analysis using cytoplasmic proteins isolated from the adipocytes treated with PKC α antisense oligomers and LPL 3′UTR transcript (LPL 3′UTR 1512-1640). Using a LPL mRNA affinity column we have separated and identified the components involved in this interaction to be, the catalytic and the regulatory subunits of PKA (Cα and RII β), and AKAP 121. The data indicated that the RNA inhibitory complex consisted of the same RNA binding proteins which have been identified by us previously as mediators of LPL translational inhibition by epinephrine. We therefore examined whether PKC α depletion was activating PKA. PKC depletion by prolonged TPA treatment (16h) or PKC α antisense oligomers increased PKA activity in 3T3 F442A adipocytes, and this activation was comparable to PKA activation following 2h of epinephrine treatment. [ABSTRACT FROM AUTHOR]

Lipid accumulation in liver and skeletal muscle contributes to co-morbidities associated with diabetes and obesity. We made a transgenic mouse in which the adiponectin (Adipoq) promoter drives expression of lipoprotein lipase (LPL) in adipocytes to potentially increase adipose tissue lipid storage. These mice (Adipoq-LPL) have improved glucose and insulin tolerance as well as increased energy expenditure when challenged with a high fat diet (HFD). To identify the mechanism(s) involved, we determined whether the Adipoq-LPL mice diverted dietary lipid to adipose tissue to reduce peripheral lipotoxicity, but we found no evidence for this. Instead, characterization of the adipose tissue of the male mice after HFD challenge revealed that the mRNA levels of peroxisome proliferator-activated receptor-γ (PPARγ) and a number of PPARγ-regulated genes were higher in the epididymal fat pads of Adipoq-LPL mice than control mice. This included adiponectin, whose mRNA levels were increased, leading to increased adiponectin serum levels in the Adipoq-LPL mice. In many respects, the adipose phenotype of these animals resembles thiazolidinedione treatment except for one important difference, the Adipoq-LPL mice did not gain more fat mass on HFD than control mice and did not have increased expression of genes in adipose such as glycerol kinase, which are induced by high affinity PPAR agonists. Rather, there was selective induction of PPARγ-regulated genes such as adiponectin in the adipose of the Adipoq-LPL mice, suggesting that increasing adipose tissue LPL improves glucose metabolism in diet-induced obesity by improving the adipose tissue phenotype. Adipoq-LPL mice also have increased energy expenditure. [ABSTRACT FROM AUTHOR]

The serotonin transporter (SERT) on the plasma membrane is the major mechanism for the clearance of plasma serotonin (5-hydroxytryptamine (5HT)). The uptake rates of cells depend on the density of SERT molecules on the plasma membrane. Interestingly, the number of SERT molecules on the platelet surface is down-regulated when plasma 5HT ([5HT]ex) is elevated. It is well reported that stimulation of cells with high [5HT]ex induces transamidation of a small GTPase, Rab4. Modification with 5HT stabilizes Rab4 in its active, GTP-bound form, Rab4-GTP. Although investigating the mechanism by which elevated plasma 5HT level down-regulates the density of SERT molecules on the plasma membrane, we studied Rab4 and SERT in heterologous and platelet expression systems. Our data demonstrate that, in response to elevated [5HT]ex, Rab4-GTP co-localizes with and binds to SERT. The association of SERT with Rab4-GTP depends on: (i) 5HT modification and (ii) the GTP-binding ability of Rab4. Their association retains transporter molecules intracellularly. Furthermore, we mapped the Rab4-SERT association domain to amino acids 616-624 in the cytoplasmic tail of SERT. This finding provides an explanation for the role of the C terminus in the localization and trafficking of SERT via Rab4 in a plasma 5HT-dependent manner. Therefore, we propose that elevated [5HT]ex "paralyzes" the translocation of SERT from intracellular locations to the plasma membrane by con- trolling transamidation and Rab4-GTP formation. [ABSTRACT FROM AUTHOR]

Context and Objective: Stearoyl-coenzyme A desaturase (SCD1) is the rate-limiting enzyme that converts palmitoyl- and stearoyl-coenzyme A to palmitoleoyl- and oleoyl-cownzyme A, respectively. SCD-deficient mice are protected from obesity, and the ob/ob mouse has high levels of SCD. This study was designed to better characterize SCD1 gene and protein expression in humans with varying insulin sensitivity., Design, Participants, and Setting: In a university hospital clinical research center setting, SCD1 gene expression was measured in sc adipose and vastus lateralis muscle of 86 nondiabetic subjects; 10 wk of pioglitazone (45 mg daily) and metformin (1000 mg twice daily) treatment were assessed in 36 impaired glucose-tolerant subjects. Adipocytes were treated with pioglitazone, and SCD1 expression was attenuated with small interfering RNA (siRNA) to examine other adipocyte genes., Results: There was no significant relationship between adipose or muscle SCD1 mRNA and either body mass index or insulin sensitivity. After pioglitazone (but not metformin) treatment, there was a 2-fold increase in SCD1 mRNA and protein in adipose tissue. Pioglitazone also increased SCD1 in vitro. There were significant positive correlations between SCD1 and peroxisomal proliferator-activated receptor gamma (PPARgamma) as well as other PPARgamma-responsive genes, including lipin-beta, AGPAT2, RBP4, adiponectin receptors, CD68, and MCP1. When SCD1 expression was inhibited with a siRNA, lipin-beta, AGPAT2, and the adiponectin R2 receptor expression were decreased, and adipocyte MCP-1 was increased., Conclusions: SCD1 is closely linked to PPARgamma expression in humans, and is increased by PPARgamma agonists. The change in expression of some downstream PPARgamma targets after SCD1 knockdown suggests that PPARgamma up-regulation of SCD1 leads to increased lipogenesis and potentiation of adiponectin signaling.

Adipose LPL (lipoprotein lipase) plays an important role in regulating plasma triacylglycerols and lipid metabolism. We have previously demonstrated that PKCalpha (protein kinase Calpha) depletion inhibits LPL translation in 3T3-F442A adipocytes. Using in vitro translation experiments, the minimum essential region on the 3'UTR (3'-untranslated region) of LPL mRNA required for the inhibition of translation was identified as the proximal 39 nt. These results were confirmed by RNase protection analysis using cytoplasmic proteins isolated from the adipocytes treated with PKCalpha antisense oligomers and the LPL 3'UTR transcript (LPL 3'UTR nt: 1512-1640). The protein components involved in this RNA-binding interaction from PKCalpha depletion were passed through an affinity column containing a sequence of the LPL 3'UTR and, after Western blotting, the RNA-binding proteins were identified as the catalytic and the regulatory subunits of PKA (protein kinase A), Calpha and RIIbeta, and AKAP (A-kinase-anchoring protein) 121. This RNA inhibitory complex consisted of the same RNA-binding proteins that have been identified previously as mediators of LPL translational inhibition by PKA activation, suggesting that PKCalpha depletion inhibits LPL translation through PKA activation. In additional experiments, PKC depletion by prolonged PMA treatment or PKCalpha antisense oligomers resulted in an increase in PKA activity in 3T3-F442A adipocytes, comparable with PKA activation with adrenaline (epinephrine) treatment. These results demonstrate that LPL translational inhibition occurs through an RNA-binding complex involving PKA subunits and AKAP121, and this complex can be activated either through traditional PKA activation methods or through the depletion of PKCalpha.

Acyl-coenzyme A:diacylglycerol transferase (DGAT), fatty acid synthetase (FAS), and LPL are three enzymes important in adipose tissue triglyceride accumulation. To study the relationship of DGAT1, FAS, and LPL with insulin, we examined adipose mRNA expression of these genes in subjects with a wide range of insulin sensitivity (SI). DGAT1 and FAS (but not LPL) expression were strongly correlated with SI. In addition, the expression of DGAT1 and FAS (but not LPL) were higher in normal glucose-tolerant subjects compared with subjects with impaired glucose tolerance (IGT) (P < 0.005). To study the effects of insulin sensitizers, subjects with IGT were treated with pioglitazone or metformin for 10 weeks, and lipogenic enzymes were measured in adipose tissue. After pioglitazone treatment, DGAT1 expression was increased by 33 +/- 10% (P < 0.05) and FAS expression increased by 63 +/- 8% (P < 0.05); however, LPL expression was not altered. DGAT1, FAS, and LPL mRNA expression were not significantly changed after metformin treatment. The treatment of mice with rosiglitazone also resulted in an increase in adipose expression of DGAT1 by 2- to 3-fold, as did the treatment of 3T3 F442A adipocytes in vitro with thiazolidinediones. These data support a more global concept suggesting that adipose lipid storage functions to prevent peripheral lipotoxicity.